Proteins having pneumococcal capsule degrading activity and methods of use

ABSTRACT

Provided herein is a protein, referred to as a Pn3Pase protein, that degrades the capsular polysaccharide of serotype 3 Streptococcus pneumoniae. The disclosure includes a genetically modified cell that includes a Pn3Pase protein, and compositions that include the protein, the polynucleotide encoding the protein, the genetically modified cell, or a combination thereof. Also provided are methods for using a Pn3Pase protein, including methods for contacting a S. pneumoniae having a type III capsular polysaccharide with a Pn3Pase protein, increasing deposition of at least one complement component on the surface of a S. pneumoniae, treating an infection in a subject, treating a symptom in a subject, decreasing colonization of a subject by S. pneumoniae, or a combination thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 16/636,503, filed Feb. 2, 2020, which is the § 371U.S. National Stage of International Application No. PCT/US2018/046521,filed Aug. 13, 2018, which claims the benefit of U.S. ProvisionalApplication Ser. No. 62/545,094, filed Aug. 14, 2017, and U.S.Provisional Application Ser. No. 62/652,046, filed Apr. 3, 2018, thedisclosures of which are incorporated by reference herein in theirentireties.

GOVERNMENT FUNDING

This invention was made with government support under 1R01AI123383-01A1,awarded by the National Institutes of Health. The government has certainrights in the invention.

SEQUENCE LISTING

This application contains a Sequence Listing electronically submittedvia Patent Center to the United States Patent and Trademark Office as anXML file entitled “0235-000271US02.xml” having a size of 44 kilobytesand created on May 17, 2023. The information contained in the SequenceListing is incorporated by reference herein.

BACKGROUND

Diseases caused by Streptococcus pneumoniae (Spn) have long been a majorthreat to human health with alarming mortality rates. One disease,pneumonia, affects approximately 450 million people globally (7% of thepopulation) and results in about 4 million deaths per year. Preventionof pneumococcal disease relies today on vaccination of the susceptiblepopulation. Pneumococcal vaccines are made empirically and arevariably/poorly immunogenic especially among young children, theelderly, and immunocompromised individuals. The mainstay of drug therapyfor pneumococcal disease is antibiotic treatment; however, widespreaduse of antibiotics against S. pneumoniae has led to spread of drugresistance pneumococcal strains. In 2015, according to CDC, pneumococcalbacteria were resistant to one or more antibiotics in 30% of the cases.

S. pneumoniae has over 90 unique capsular serotypes, each differing inmonosaccharide composition and linkage, as well as other modificationssuch as acetylation. While conjugate vaccines have been effective forpreventing carriage and disease caused by most S. pneumoniae serotypesincluded in the vaccines, the exception has been serotype 3.

SUMMARY

Described herein is the identification and cloning of a Pn3Pase geneexpressed by a Paenibacillus strain. The Pn3Pase enzyme degrades thecapsular polysaccharide of the S. pneumoniae serotype 3, which is amongthe most virulent serotypes with high morbidity and mortality rates.Capsular polysaccharide is the major virulence factor of S. pneumoniae(Spn), and most unencapsulated bacteria are not infective. The enzymesuccessfully degrades the capsular polysaccharide on the surface of thelive bacterium. Moreover, the bacterium is susceptible to phagocytosisin the presence of the enzyme.

Provided herein is a genetically modified cell that includes a maturePn3Pase protein. In one embodiment, the genetically modified cellincludes a polynucleotide including a coding region, where the codingregion includes a nucleotide sequence encoding a mature Pn3Pase protein.The protein can include an amino acid sequence having at least 80%identity with an amino acid sequence selected from SEQ ID NO:2, wherethe amino terminal amino acid is selected from any one of residues 2 to64 of SEQ ID NO:2 and the carboxy terminal amino acid is residue 1545 ofSEQ ID NO:2. In one embodiment, the protein includes an amino acidsequence having at least 80% identity with amino acids 41-1545 of SEQ IDNO:2. In one embodiment, the genetically modified cell can be aeukaryotic cell, such as a mammalian cell, a yeast cell, or an insectcell. In one embodiment, the genetically modified cell can be aprokaryotic cell, such as E. coli. In one embodiment, the proteinincludes a heterologous amino acid sequence, such as a tag.

Also provided herein are compositions. In one embodiment, a compositionincludes the genetically modified cell. In one embodiment, a compositionincludes an isolated, optionally purified, mature P3nPase protein. Inone embodiment, a composition includes an isolated polynucleotide, wherethe polynucleotide includes a coding region encoding a mature Pn3Paseprotein. The mature P3nPase protein can include an amino acid sequencehaving at least 80% identity with an amino acid sequence selected fromSEQ ID NO:2, where the amino terminal amino acid is selected fromresidues 2 to 64 of SEQ ID NO:2 and the carboxy terminal amino acid isresidue 1545 of SEQ ID NO:2. In one embodiment, the protein includes anamino acid sequence having at least 80% identity with amino acids41-1545 of SEQ ID NO:2. In one embodiment, a composition furtherincludes a pharmaceutically acceptable carrier.

Also provided herein are methods. In one embodiment, a method includesincubating a cell under conditions suitable for expression of a proteinhaving Pn3Pase activity. The cell can include a polynucleotide includinga coding region, where the coding region includes a nucleotide sequenceencoding a mature Pn3Pase protein, where the protein has Pn3Paseactivity, and where the cell expresses the mature Pn3Pase protein undersuitable conditions. The protein can include an amino acid sequencehaving at least 80% identity with an amino acid sequence selected fromSEQ ID NO:2, where the amino terminal amino acid is selected from anyone of residues 2 to 64 of SEQ ID NO:2 and the carboxy terminal aminoacid is residue 1545 of SEQ ID NO:2. In one embodiment, the proteinincludes an amino acid sequence having at least 80% identity with aminoacids 41-1545 of SEQ ID NO:2. The cell can be a wild-type cell, e.g., aPaenibacillus strain, such as Paenibacillus sp. 32352, or a geneticallymodified cell where the polynucleotide encoding the is an exogenouspolynucleotide. The method can further include isolating or purifyingthe protein. In one embodiment, the cell can be a eukaryotic cell, suchas a mammalian cell, a yeast cell, or an insect cell. In one embodiment,the genetically modified cell can be a prokaryotic cell, such as E.coli. In one embodiment, the protein includes a heterologous amino acidsequence, such as a tag.

In one embodiment, a method includes contacting a Streptococcuspneumoniae including a type III capsular polysaccharide with a maturePn3Pase protein, where the contacting is under conditions suitable forenzymatic hydrolysis of type III capsular polysaccharide, where theamount of type III capsular polysaccharide on the surface of the S.pneumoniae is reduced compared to the S. pneumoniae that is notcontacted with the Pn3Pase protein. In another embodiment, a method isfor increasing deposition of at least one complement component on thesurface of a Streptococcus pneumoniae. In one embodiment, the method caninclude contacting a Streptococcus pneumoniae including a type IIIcapsular polysaccharide with a mature Pn3Pase protein, where thedeposition of at least one complement component on the surface of the S.pneumoniae is increased compared to the S. pneumoniae that is notcontacted with the Pn3Pase protein. The protein can include an aminoacid sequence having at least 80% identity with an amino acid sequenceselected from SEQ ID NO:2, where the amino terminal amino acid isselected from any one of residues 2 to 64 of SEQ ID NO:2 and the carboxyterminal amino acid is residue 1545 of SEQ ID NO:2. In one embodiment,The protein includes an amino acid sequence having at least 80% identitywith amino acids 41-1545 of SEQ ID NO:2. In one embodiment, the S.pneumoniae is present in conditions suitable for replication of the S.pneumoniae. In one embodiment, the mature Pn3Pase protein is an isolatedPn3Pase protein. In one embodiment, the contacting includes exposing theS. pneumoniae to a genetically modified cell that expresses the maturePn3Pase protein. In one embodiment, the S. pneumoniae has increasedsusceptibility to phagocytosis by macrophages, increasedcomplement-mediated killing by neutrophils, or a combination thereof,compared to the S. pneumoniae that is not contacted with the Pn3Paseprotein. In one embodiment, the S. pneumoniae is present in a subject.

In one embodiment, a method is for treating an infection in a subjectand includes administering an effective amount of a composition thatincludes a mature Pn3Pase protein having Pn3Pase activity to a subjecthaving or at risk of having an infection caused by a serotype 3 S.pneumoniae. In one embodiment, the method is for treating a symptom in asubject and includes administering an effective amount of a compositionincluding a mature Pn3Pase protein having Pn3Pase activity to a subjecthaving or at risk of having an infection caused by a serotype 3 S.pneumoniae. In one embodiment, a method is for decreasing colonizationin a subject and includes administering an effective amount of acomposition including a including a mature Pn3Pase protein havingP3nPase activity to a subject colonized by or at risk of being colonizedby a serotype 3 S. pneumoniae. The protein can include an amino acidsequence having at least 80% identity with an amino acid sequenceselected from SEQ ID NO:2, where the amino terminal amino acid isselected from any one of residues 2 to 64 of SEQ ID NO:2 and the carboxyterminal amino acid is residue 1545 of SEQ ID NO:2. In one embodiment,the protein includes an amino acid sequence having at least 80% identitywith amino acids 41-1545 of SEQ ID NO:2. In one embodiment, the subjectis a human.

Also provided is a mature Pn3Pase protein disclosed herein for use intherapy, mature Pn3Pase protein disclosed herein for use as amedicament, and a mature Pn3Pase protein disclosed herein for use in thetreatment of a condition.

Further provided is the use of a mature Pn3Pase protein disclosed hereinfor preparation of a medicament for the treatment of pneumonia,pneumococcal meningitis, otitis media, bacteremia, sepsis, or acombination thereof; a composition including a mature P3nPase proteindescribed herein for use in treating or preventing an infection or asymptom caused by a serotype 3 S. pneumoniae; and a protein,composition, or method including one or more features described herein.

As used herein, “genetically modified cell” refers to a cell into whichhas been introduced an exogenous polynucleotide, such as an expressionvector. For example, a cell is a genetically modified cell by virtue ofintroduction into a suitable cell of an exogenous polynucleotide that isforeign to the cell. “Genetically modified cell” also refers to a cellthat has been genetically manipulated such that endogenous nucleotideshave been altered. For example, a cell is a genetically modified cell byvirtue of introduction into a suitable cell of an alteration ofendogenous nucleotides. An example of a genetically modified cell is onehaving an altered regulatory sequence, such as a promoter, to result inincreased or decreased expression of an operably linked endogenouscoding region.

As used herein, the term “protein” refers broadly to a polymer of two ormore amino acids joined together by peptide bonds. The term “protein”also includes molecules which contain more than one protein joined by adisulfide bond, or complexes of proteins that are joined together,covalently or noncovalently, as multimers (e.g., dimers, tetramers).Thus, the terms peptide, oligopeptide, enzyme, and polypeptide are allincluded within the definition of protein and these terms are usedinterchangeably. It should be understood that these terms do not connotea specific length of a polymer of amino acids, nor are they intended toimply or distinguish whether the protein is produced using recombinanttechniques, chemical or enzymatic synthesis, or is naturally occurring.

As used herein, a “mature protein” refers to a protein having an aminoacid sequence that is identical to or having structural similarity witha protein that has been processed after translation to result in itsfinal form. Post-translation processing can include N-terminalprocessing or C-terminal truncation. As used herein, a “preprocessedprotein” refers to a protein that has been translated from a mRNA andhas not undergone post-translation processing to result in a matureprotein.

As used herein, an “isolated” substance, for instance a protein, is onethat has been removed from its natural environment, produced usingrecombinant techniques, or chemically or enzymatically synthesized. Forinstance, a protein or a polynucleotide can be isolated. Preferably, asubstance is purified, i.e., is at least 60% free, preferably at least75% free, and most preferably at least 90% free from other componentswith which they are naturally associated.

As used herein, the term “polynucleotide” refers to a polymeric form ofnucleotides of any length, either ribonucleotides or deoxynucleotides,and includes both double- and single-stranded RNA and DNA. Apolynucleotide can be obtained directly from a natural source, or can beprepared with the aid of recombinant, enzymatic, or chemical techniques.A polynucleotide can be linear or circular in topology. A polynucleotidemay be, for example, a portion of a vector, such as an expression orcloning vector, or a fragment. A polynucleotide may include nucleotidesequences having different functions, including, for instance, codingregions, and non-coding regions such as regulatory regions.

As used herein, a “detectable moiety” or “label” is a molecule that isdetectable, either directly or indirectly, by spectroscopic,photochemical, biochemical, immunochemical, or chemical means. Forexample, useful labels include ³²P, fluorescent dyes, electron-densereagents, enzymes and their substrates (e.g., as commonly used inenzyme-linked immunoassays, e.g., alkaline phosphatase and horse radishperoxidase), biotin-streptavidin, digoxigenin, proteins such asantibodies, or haptens and proteins for which antisera or monoclonalantibodies are available. The label or detectable moiety is typicallybound, either covalently, through a linker or chemical bound, or throughionic, van der Waals or hydrogen bonds to the molecule to be detected.

As used herein, the terms “coding region” and “coding sequence” are usedinterchangeably and refer to a nucleotide sequence that encodes aprotein and, when placed under the control of appropriate regulatorysequences expresses the encoded protein. The boundaries of a codingregion are generally determined by a translation start codon at its 5′end and a translation stop codon at its 3′ end. A “regulatory sequence”is a nucleotide sequence that regulates expression of a coding sequenceto which it is operably linked. Non-limiting examples of regulatorysequences include promoters, enhancers, transcription initiation sites,translation start sites, translation stop sites, and transcriptionterminators. The term “operably linked” refers to a juxtaposition ofcomponents such that they are in a relationship permitting them tofunction in their intended manner. A regulatory sequence is “operablylinked” to a coding region when it is joined in such a way thatexpression of the coding region is achieved under conditions compatiblewith the regulatory sequence.

A polynucleotide that includes a coding region may include heterologousnucleotides that flank one or both sides of the coding region. As usedherein, “heterologous nucleotides” refer to nucleotides that are notnormally present flanking a coding region that is present in a wild-typecell. Thus, a polynucleotide that includes a coding region andheterologous nucleotides is not a naturally occurring molecule. Forinstance, a coding region present in a wild-type microbe and encoding aPn3Pase protein is flanked by homologous sequences, and any othernucleotide sequence flanking the coding region is considered to beheterologous. Examples of heterologous nucleotides include, but are notlimited to regulatory sequences. Typically, heterologous nucleotides arepresent in a polynucleotide described herein through the use of standardgenetic and/or recombinant methodologies well known to one skilled inthe art. A polynucleotide described herein may be included in a suitablevector.

A protein described herein may include heterologous amino acids presentat the N-terminus, the C-terminus, or a combination thereof. As usedherein, “heterologous amino acids” refer to amino acids that are notnormally present flanking a protein that is naturally present in awild-type cell. Thus, a protein that includes heterologous amino acidsis not a naturally occurring molecule. For instance, a naturallyoccurring Pn3Pase protein present in a wild-type microbe does not haveadditional amino acids at either the N-terminal end or the C-terminalend, and any other amino acids present at the N-terminal end or theC-terminal end are considered to be heterologous. Examples ofheterologous amino acid sequences are described herein, and include, butare not limited to affinity purification tags. Typically, heterologousamino acids are present in a protein described herein through the use ofstandard genetic and/or recombinant methodologies well known to oneskilled in the art.

As used herein, an “exogenous polynucleotide” refers to a polynucleotidethat is not normally or naturally found in a cell. As used herein, theterm “endogenous polynucleotide” refers to a polynucleotide that isnormally or naturally found in a cell. An “endogenous polynucleotide” isalso referred to as a “native polynucleotide.”

The terms “complement” and “complementary” as used herein, refer to theability of two single stranded polynucleotides to base pair with eachother, where an adenine on one strand of a polynucleotide will base pairto a thymine or uracil on a strand of a second polynucleotide and acytosine on one strand of a polynucleotide will base pair to a guanineon a strand of a second polynucleotide. Two polynucleotides arecomplementary to each other when a nucleotide sequence in onepolynucleotide can base pair with a nucleotide sequence in a secondpolynucleotide. For instance, 5′-ATGC and 5′-GCAT are complementary. Theterm “substantial complement” and cognates thereof as used herein referto a polynucleotide that is capable of selectively hybridizing to aspecified polynucleotide under stringent hybridization conditions.Stringent hybridization can take place under a number of pH, salt, andtemperature conditions. The pH can vary from 6 to 9, preferably 6.8 to8.5. The salt concentration can vary from 0.15 M sodium to 0.9 M sodium,and other cations can be used as long as the ionic strength isequivalent to that specified for sodium. The temperature of thehybridization reaction can vary from 30° C. to 80° C., preferably from45° C. to 70° C. Additionally, other compounds can be added to ahybridization reaction to promote specific hybridization at lowertemperatures, such as at or approaching room temperature. Among thecompounds contemplated for lowering the temperature requirements isformamide. Thus, a polynucleotide is typically substantiallycomplementary to a second polynucleotide if hybridization occurs betweenthe polynucleotide and the second polynucleotide. As used herein,“specific hybridization” refers to hybridization between twopolynucleotides under stringent hybridization conditions.

In the comparison of two amino acid sequences, structural similarity maybe referred to by percent “identity” or may be referred to by percent“similarity.” “Identity” refers to the presence of identical aminoacids. “Similarity” refers to the presence of not only identical aminoacids but also the presence of conservative substitutions. The sequencesimilarity between two proteins is determined by aligning the residuesof the two proteins (e.g., a candidate amino acid sequence and areference amino acid sequence, such as a mature Pn3Pase protein, e.g.,amino acids 41-1545 of SEQ ID NO:2) to optimize the number of identicalamino acids along the lengths of their sequences; gaps in either or bothsequences are permitted in making the alignment in order to optimize thenumber of shared amino acids, although the amino acids in each sequencemust nonetheless remain in their proper order. Sequence similarity maybe determined, for example, using sequence techniques such as theBESTFIT algorithm in the GCG package (Madison WI), or the Blastp programof the BLAST 2 search algorithm, as described by Tatusova, et al. (FEMSMicrobiol Lett 1999, 174:247-250), and available through the World WideWeb, for instance at the internet site maintained by the National Centerfor Biotechnology Information, National Institutes of Health.Preferably, sequence similarity between two amino acid sequences isdetermined using the Blastp program of the BLAST 2 search algorithm.Preferably, the default values for all BLAST 2 search parameters areused. In the comparison of two amino acid sequences using the BLASTsearch algorithm, structural similarity is referred to as “identities.”Thus, reference to a protein described herein, such as a mature Pn3Paseprotein, e.g., amino acids 41-1545 of SEQ ID NO:2, can include a proteinwith at least 80% identity, at least 81% identity, at least 82%identity, at least 83% identity, at least 84% identity, at least 85%identity, at least 86% identity, at least 87% identity, at least 88%identity, at least 89% identity, at least 90% identity, at least 91%identity, at least 92% identity, at least 93% identity, at least 94%identity, at least 95% identity, at least 96% identity, at least 97%identity, at least 98% identity, or at least 99% identity with thereference protein. Alternatively, reference to a protein describedherein, such as a mature Pn3Pase protein, e.g., amino acids 41-1545 ofSEQ ID NO:2, can include a protein with at least 80% similarity, atleast 81% similarity, at least 82% similarity, at least 83% similarity,at least 84% similarity, at least 85% similarity, at least 86%similarity, at least 87% similarity, at least 88% similarity, at least89% similarity, at least 90% similarity, at least 91% similarity, atleast 92% similarity, at least 93% similarity, at least 94% similarity,at least 95% similarity, at least 96% similarity, at least 97%similarity, at least 98% similarity, or at least 99% similarity with thereference protein.

The sequence similarity between two polynucleotides is determined byaligning the residues of the two polynucleotides (e.g., a candidatenucleotide sequence and a reference nucleotide sequence encoding amature Pn3Pase protein) to optimize the number of identical nucleotidesalong the lengths of their sequences; gaps in either or both sequencesare permitted in making the alignment in order to optimize the number ofshared nucleotides, although the nucleotides in each sequence mustnonetheless remain in their proper order. Sequence similarity may bedetermined, for example, using sequence techniques such as GCG FastA(Genetics Computer Group, Madison, Wisconsin), MacVector 4.5 (Kodak/IBIsoftware package) or other suitable sequencing programs or methods knownin the art. Preferably, sequence similarity between two nucleotidesequences is determined using the Blastn program of the BLAST 2 searchalgorithm, as described by Tatusova, et al. (1999, FEMS Microbiol Lett.,174:247-250), and available through the World Wide Web, for instance atthe internet site maintained by the National Center for BiotechnologyInformation, National Institutes of Health. Preferably, the defaultvalues for all BLAST 2 search parameters are used. In the comparison oftwo nucleotide sequences using the BLAST search algorithm, sequencesimilarity is referred to as “identities.” The sequence similarity istypically at least 50% identity, at least 55% identity, at least 60%identity, at least 65% identity, at least 70% identity, at least 75%identity, at least 80% identity, at least 81% identity, at least 82%identity, at least 83% identity, at least 84% identity, at least 85%identity, at least 86% identity, at least 87% identity, at least 88%identity, at least 89% identity, at least 90% identity, at least 91%identity, at least 92% identity, at least 93% identity, at least 94%identity, at least 95% identity, at least 96% identity, at least 97%identity, at least 98% identity, or at least 99% identity.

As used herein, “in vitro” refers to an artificial environment and toprocesses or reactions that occur within an artificial environment. Invitro environments can include, but are not limited to, a test tube. Asused herein, the term “in vivo” refers to a natural environment that iswithin the body of a subject.

Conditions that “allow” an event to occur or conditions that are“suitable” for an event to occur, such as an enzymatic reaction, or“suitable” conditions are conditions that do not prevent such eventsfrom occurring. Thus, these conditions permit, enhance, facilitate,and/or are conducive to the event.

The term “and/or” means one or all of the listed elements or acombination of any two or more of the listed elements.

The words “preferred” and “preferably” refer to embodiments of thedisclosure that may afford certain benefits, under certaincircumstances. However, other embodiments may also be preferred, underthe same or other circumstances. Furthermore, the recitation of one ormore preferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the disclosure.

The terms “comprises” and variations thereof do not have a limitingmeaning where these terms appear in the description and claims.

It is understood that wherever embodiments are described herein with thelanguage “include,” “includes,” or “including,” and the like, otherwiseanalogous embodiments described in terms of “consisting of” and/or“consisting essentially of” are also provided.

Unless otherwise specified, “a,” “an,” “the,” and “at least one” areused interchangeably and mean one or more than one.

Also herein, the recitations of numerical ranges by endpoints includeall numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc.).

For any method disclosed herein that includes discrete steps, the stepsmay be conducted in any feasible order. And, as appropriate, anycombination of two or more steps may be conducted simultaneously.

Reference throughout this specification to “one embodiment,” “anembodiment,” “certain embodiments,” or “some embodiments,” etc., meansthat a particular feature, configuration, composition, or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the disclosure. Thus, the appearances of such phrases invarious places throughout this specification are not necessarilyreferring to the same embodiment of the disclosure. Furthermore, theparticular features, configurations, compositions, or characteristicsmay be combined in any suitable manner in one or more embodiments.

The above summary of the present disclosure is not intended to describeeach disclosed embodiment or every implementation of the presentdisclosure. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the application,guidance is provided through lists of examples, which examples can beused in various combinations. In each instance, the recited list servesonly as a representative group and should not be interpreted as anexclusive list.

In the description herein particular embodiments may be described inisolation for clarity. Unless otherwise expressly specified that thefeatures of a particular embodiment are incompatible with the featuresof another embodiment, certain embodiments can include a combination ofcompatible features described herein in connection with one or moreembodiments.

BRIEF DESCRIPTION OF THE FIGURES

The following detailed description of illustrative embodiments of thepresent disclosure may be best understood when read in conjunction withthe following drawings.

FIG. 1 shows a repeating unit structure of Streptococcus pneumoniae(Spn) serotype 3 capsular polysaccharide.

FIG. 2 shows an amino acid alignment of seven glycoside hydrolases fromPaenibacillus. WP_079915027.1 (SEQ ID NO:2), WP_113018908.1 (SEQ IDNO:4), WP_068663733.1 (SEQ ID NO:5), KRE82476.1 (SEQ ID NO:6),WP_082593776.1 (SEQ ID NO:7), WP_056617367.1 (SEQ ID NO:8), andWP_028553222.1 (SEQ ID NO:9).

FIGS. 3A-3D show culture of Paenibacillus sp. 32352 in minimal mediumwith Pn3P as carbon source. (FIG. 3A) Growth of Paenibacillus sp. 32352(Pbac) on minimal medium plus 2% (w/v) glucose, Pn3P, cellulose, ornothing as carbon source. (FIG. 3B) SDS-PAGE coomassie blue stained gelof concentrated culture supernatant of Pbac grown on glucose (lane 1) orPn3P (lane 2). (FIG. 3C) Proposed locus of Pn3P utilization organizationbased on the Rapid Annotation Server (Aziz et al. 2008, BMC Genomics 9,75) (RAST) annotation. (FIG. 3D) Real-Time PCR of select genes withinputative locus in Pbac grown on 2% glucose or Pn3P, shown as fold changein expression in Pn3P culture versus glucose culture.

FIGS. 4A-4E show Pn3Pase identification and domain schematic of primaryamino acid sequence. (FIG. 4A) Amino acid sequence (SEQ ID NO:2) andaccession of Pn3Pase. (FIG. 4B) Separation of recombinant Pn3Pasedepolymerized tritium radioisotope labeled Pn3P by size exclusionchromatography, measured by counts per minute in each 1 ml fraction.(FIG. 4C) Dot blot of recombinant Pn3Pase depolymerized cold Pn3P probedwith Pn3P monoclonal antibody. (FIG. 4D) Schematic of predicted domainsof Pn3Pase by InterPro. (FIG. 4E) A nucleotide sequence (SEQ ID NO:1)encoding a Pn3Pase.

FIGS. 5A-5C show identification of oligosaccharide products byelectrospray ionization mass spectrometry. Separation of oligosaccharideproducts (FIG. 5A) and mass spectra of the tetrasaccharide (FIG. 5B) andthe hexasaccharide (FIG. 5C). Experimental molecular weights and theaccuracies are shown in Table 1.

FIGS. 6A-6F show Nuclear Magnetic Resonance characterization ofoligosaccharide products. 1H NMR spectrum (FIG. 6A), 2D HSQC NMRspectrum (FIG. 6B), and 2D COSY NMR spectrum (FIG. 6C) of the Pn3tetrasaccharide. 1H NMR spectrum (FIG. 6D), 2D HSQC NMR spectrum (FIG.6E), and 2D COSY NMR spectrum (FIG. 6F) of the Pn3 hexasaccharide.

FIGS. 7A-7C show activity assessment of Pn3Pase. (FIG. 7A) Time courseassay of recombinant Pn3Pase depolymerized tritium radioisotope labeledPn3P separated by size exclusion chromatography, measured by counts perminute in each 1 ml fraction. (FIG. 7B) Optimization of recombinantPn3Pase in three different buffer conditions, measured by concentrationof glucuronic acid reducing end generated in μg/ml. (FIG. 7C) Metaldependence determination with Mg2+ and Ca2+ reaction supplements,measured by concentration of glucuronic acid reducing end generated inμg/ml. Statistical significance was determined with the two-tailedStudent t-test ** P<0.01

FIGS. 8A-8D show Pn3Pase treatment of live, growing type 3 Streptococcuspneumoniae WU2 strain. (FIG. 8A) Growth curve of WU2 in THY broth in thepresence of 100 ug/ml Pn3Pase following the OD at 600 nm. (FIG. 8B-8C)Competition ELISA in which acapsular WU2, or Pn3Pase treated WU2 wasused to compete for Pn3P specific antibody binding to Pn3P coated ELISAplate. Data is presented as percent inhibition of antibody binding.(FIG. 8D) Transmission Electron Microscopy of WU2 (upper left),acapsular derivative (upper right), and representative Pn3Pase treatedWU2 (bottom panels). Statistical significance was determined with thetwo-tailed Student t test ** P<0.01, *** P<0.001.

FIGS. 9A-3B show the effects of Pn3Pase treatment on type 3 Spnviability. (FIG. 9A) Growth curve of WU2 in THY broth in the presence of100 μg/ml Pn3Pase following the OD at 600 nm. (FIG. 9B) WU2 survival inPBS in the presence of 100 μg/ml active or heat inactivated Pn3Pase.

FIGS. 10A-10E show depleting the capsule on live type 3 Spn by Pn3Pasetreatment. (FIGS. 10A-B) Competition ELISA in which acapsular WU2, WU2treated with heat-inactivated Pn3Pase, or WU2 treated with Pn3Pase attwo different concentrations (2 μg/ml or 10 μg/ml) were used to competefor Pn3P specific antibody binding to Pn3P coated ELISA plate. Data ispresented as percent inhibition of antibody binding. Statisticalsignificance was determined with the two-tailed Student t test **P<0.01, *** P<0.001. Transmission electron microscopy images of WU2 mocktreated with 2 μg/ml heat-inactivated Pn3Pase (FIG. 10C), acapsular WU2strain (FIG. 10D) and 2 μg/ml active-Pn3Pase treated WU2 (FIG. 10E).Bottom panels are imaged at 15000× direct magnification and top panelsare imaged at 10000× direct magnification (FIGS. 10C-10E).

FIGS. 11A-11C show macrophage uptake of Pn3Pase treated type 3 Spn.(FIG. 11A) RAW 264.7 macrophage containing fluorescent streptococcifollowing Heat inactivated (left) or active (right) Pn3Pase treatment.Streptococci, CFSE, green. Macrophage, Biotinylated-WGA,Streptavidin-APC, red. (FIG. 11B) Influence of Pn3Pase treatment andcomplement on macrophage uptake of CFSE labeled Spn quantified by flowcytometry. (FIG. 11C) Pn3Pase dose dependent effect on the percent offluorescent phagocytes. Statistical significance was determined with thetwo-tailed Student t test *** P<0.001.

FIG. 12 shows effect of Pn3Pase treatment on complement deposition onSpn surface. Analysis of mouse complement deposition on Pn3Pase treatedor untreated type 3 Spn by flow Cytometry. Quantification by Meanfluorescent intensity (MFI) of FITC-A was calculated from gating ofHoechst-positive cells. Statistical significance was determined with thetwo-tailed Student t test ** P<0.01, ** * P<0.001, ns=not significant.

FIG. 13 shows effect of Pn3Pase treatment on complement mediated killingby neutrophils. Complement mediated killing capacity of differentiatedHL-60 cells on Pn3Pase treated Spn. Percent survival was calculated aseach duplicate reaction normalized to mean values obtained for controlsamples (reactions without HL60 cells, 100% survival). Statisticalsignificance was determined with the two-tailed Student t test ***P<0.001, ** P<0.01, * P<0.05, ns=not significant.

FIGS. 14A-14C show intranasal colonization with type 3 Spn. Ability ofPn3Pase treatment to reduce Spn colonization in nasopharynx of BALB/cmice. (FIG. 14A) Groups of mice were intranasally inoculated with 106log-phase bacteria in 10 μl PBS. All inocula were chased with either 50g of Pn3Pase or buffer control (10 l). Groups were dosed with the enzymeat either day 0 (WU2+Pn3Pase*1), day 0 and 3 (WU2+Pn3Pase*2), or day 0,3, and 7 (WU2+Pn3Pase*3). Bacterial load was quantified on day 10.Serial dilutions of nasal lavage fluid were plated in duplicate todetermine CFU values. (FIG. 14B) IL-6 and (FIG. 14C) TNFα concentrationsin nasal lavage fluid was determined by ELISA. Statistical significancewas determined with the two-tailed Student t test * P<0.05, ** P<0.01.

FIG. 15 shows protective ability of Pn3Pase. Assessment of ability ofPn3Pase to protect BALB/c mice from lethal challenge. Groups of micewere infected through intraperitoneal administration of 5×103 log-phasevirulent type 3 Spn. Inactivated group is heat inactivated Pn3Pase.*Shown are the effect of a single dose of 5 g or 0.5 g, administered attime 0, 12, or 24 hours post infection, n=5 mice per group.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS Proteins

Provided herein are genetically modified cells and methods for using thecells. The genetically modified cells can produce a protein havingPn3Pase activity. A protein having Pn3Pase activity is referred toherein as a Pn3Pase protein or a Pn3Pase. A protein having Pn3Paseactivity degrades type III capsular polysaccharide of Streptococcuspneumoniae (Spn). This capsular polysaccharide is also known asPneumococcal type-3 polysaccharide (Pn3P) and is expressed by serotype 3(also referred to as type 3) S. pneumoniae. The structure of Pn3P isshown in FIG. 1 .

Whether a protein has Pn3Pase activity can be determined by in vitroassays. In one embodiment, an in vitro assay is carried out as describedherein (see Example 1). Briefly, Pn3P can be labeled with a detectablemoiety, exposed to a protein being tested for Pn3Pase activity, and thereaction products resolved using a method that permits detection ofdifferences in molecular weight. Pn3P can be obtained using methods thatare known to the skilled person and routine or can be purchased fromAmerican Type Culture Collection, ATCC (Manassas, VA). Methods fordetecting changes in molecular weight of polysaccharides are known tothe skilled person and are routine.

A Pn3Pase protein can be, and in some embodiments preferably is, amature protein. In one embodiment, a Pn3Pase protein is encoded by acoding sequence obtained from a wild-type cell where the protein hasbeen processed to remove one or more amino acids from the N-terminal endto result in a mature protein, e.g., the protein lacks a signalsequence. Thus, a mature protein can lack, when compared to apreprocessed Pn3Pase protein encoded by a coding sequence obtained froma wild-type cell, at least one, at least two, at least three, at leastfour, at least five, at least six, at least seven, at least eight, atleast nine, at least ten, at least 11, at least 12, at least 13, atleast 14, at least 15, at least 16, at least 17, at least 18, at least19, at least 20, at least 21, at least 22, at least 23, at least 24, atleast 25, at least 26, at least 27, at least 28, at least 29, at least30, at least 31, at least 32, at least 33, at least 34, at least 35, atleast 36, at least 37, at least 38, at least 39, at least 40, at least41, at least 42, at least 43, at least 44, at least 45, at least 46, atleast 47, at least 48, at least 49, at least 50, at least 51, at least52, at least 53, at least 54, at least 55, at least 56, at least 57, atleast 58, at least 59, at least 60, at least 61, at least 62, or atleast 63 amino acids from the amino terminus of the preprocessedprotein. In another embodiment, a Pn3Pase protein is encoded by a codingregion that does not include nucleotides encoding amino acidscorresponding to a signal sequence.

An example of a Pn3Pase protein is depicted at SEQ ID NO:2, where thePn3Pase protein is a preprocessed protein. Examples of a mature Pn3Paseprotein include an amino acid sequence having an N-terminal amino acidthat is selected from amino acid 2-64 of SEQ ID NO:2 and the C-terminalamino acid is residue 1545 of SEQ ID NO:2. Other examples of Pn3Paseproteins include those having sequence similarity with the amino acidsequence of SEQ ID NO:2 or a mature Pn3Pase protein that includes asubset of amino acids of SEQ ID NO:2. A Pn3Pase protein having sequencesimilarity with the amino acid sequence of SEQ ID NO:2 or a portionthereof has Pn3Pase activity. A preprocessed or mature Pn3Pase proteinmay be isolated from a microbe, such as a member of the genera Bacillus,such as B. circulans. A specific example of a microbe is Bacilluscirculans Jordan strain 32352 (ATCC 14175). Data from determining thenucleotide sequence of Bacillus circulans indicates this bacteriumshould be reclassified in the genus Paenibacillus, as its nearestneighbors phylogenetically lie within this classification. Paenibacillusspecies were previously included within the Bacillus genus until theywere deemed appropriately dissimilar (Ash et al., 1993, Antonie VanLeeuwenhoek 64:253-260). Classification into the Paenibacillus genus wasconfirmed by the presence of the unique group 3 bacilli (Paenibacilli)nucleotide sequence (TCGATACCCTTGGTGCCGAAGT, SEQ ID NO:3) on 16sribosomal RNA (Ash et al., 1993, Antonie Van Leeuwenhoek 64:253-260). APn3Pase protein can also be produced using recombinant techniques, orchemically or enzymatically synthesized using routine methods. Arecombinantly-produced protein may include the entire amino acidsequence translatable from an mRNA transcript, or a portion thereof thatencodes a mature Pn3Pase protein.

The amino acid sequence of a Pn3Pase protein having sequence similarityto SEQ ID NO:2 or a portion thereof corresponding to a mature Pn3Paseprotein may include conservative substitutions of amino acids. Aconservative substitution is typically the substitution of one aminoacid for another that is a member of the same class. For example, it iswell known in the art of protein biochemistry that an amino acidbelonging to a grouping of amino acids having a particular size orcharacteristic (such as charge, hydrophobicity, and/or hydrophilicity)may generally be substituted for another amino acid withoutsubstantially altering the secondary and/or tertiary structure of aprotein. For the purposes of this disclosure, conservative amino acidsubstitutions are defined to result from exchange of amino acidsresidues from within one of the following classes of residues: Class I:Gly, Ala, Val, Leu, and Ile (representing aliphatic side chains); ClassII: Gly, Ala, Val, Leu, Ile, Ser, and Thr (representing aliphatic andaliphatic hydroxyl side chains); Class III: Tyr, Ser, and Thr(representing hydroxyl side chains); Class IV: Cys and Met (representingsulfur-containing side chains); Class V: Glu, Asp, Asn and Gln (carboxylor amide group containing side chains); Class VI: His, Arg and Lys(representing basic side chains); Class VII: Gly, Ala, Pro, Trp, Tyr,Ile, Val, Leu, Phe and Met (representing hydrophobic side chains); ClassVIII: Phe, Trp, and Tyr (representing aromatic side chains); and ClassIX: Asn and Gln (representing amide side chains). The classes are notlimited to naturally occurring amino acids, but also include artificialamino acids, such as beta or gamma amino acids and those containingnon-natural side chains, and/or other similar monomers such ashydroxyacids.

Guidance concerning how to make phenotypically silent amino acidsubstitutions is provided in Bowie et al. (1990, Science,247:1306-1310), wherein the authors indicate proteins are surprisinglytolerant of amino acid substitutions. For example, Bowie et al. disclosethat there are two main approaches for studying the tolerance of aprotein sequence to change. The first method relies on the process ofevolution, in which mutations are either accepted or rejected by naturalselection. The second approach uses genetic engineering to introduceamino acid changes at specific positions of a cloned gene and selects orscreens to identify sequences that maintain functionality. As stated bythe authors, these studies have revealed that proteins are surprisinglytolerant of amino acid substitutions. The authors further indicate whichchanges are likely to be permissive at a certain position of theprotein. For example, most buried amino acid residues require non-polarside chains, whereas few features of surface side chains are generallyconserved. Other such phenotypically silent substitutions are describedin Bowie et al, and the references cited therein.

Guidance on how to modify the amino acid sequences of proteins disclosedherein is also provided at FIG. 2 . FIG. 2 depicts a Clustal Omega aminoacid alignment of glycoside hydrolase proteins expressed byPaenibacillus. Clustal Omega is a multiple sequence alignment program(Sievers et al., 2011, Molecular Systems Biology 7: 539,doi:10.1038/msb.2011.75; Goujon et al., 2010, Nucleic acids research 38(Suppl 2):W695-9, doi:10.1093/nar/gkg313). In FIG. 2 an asterisk (*)indicates positions which have a single, fully conserved residue; acolon (:) indicates conservation between groups of strongly similarproperties, roughly equivalent to scoring >0.5 in the Gonnet PAM 250matrix; a period (.) indicates conservation between groups of weaklysimilar properties, roughly equivalent to scoring=<0.5 and >0 in theGonnet PAM 250 matrix. By reference to this figure, the skilled personcan predict which alterations to an amino acid sequence are likely tomodify enzymatic activity, as well as which alterations are unlikely tomodify enzymatic activity.

Pn3Pase proteins include conserved domains including a glycosidehydrolase, family 39 domain (amino acids 180-353 of SEQ ID NO:2),galactose-binding domain-like (amino acids 621-765 of SEQ ID NO:2), adomain of unknown function DUF1080 (amino acids 781-950 of SEQ ID NO:2)with structural similarity to an endo-1,3-1,4-beta glucanase, and aconcanavalin A-like lectin/glucanase domain (amino acids 1,209-1,348 ofSEQ ID NO:2). In one embodiment, a Pn3Pase protein has thecharacteristic of displaying slightly better activity in sodiumphosphate buffer at pH 7.2 than in MES buffer pH 6.0, and significantlyworse in Tris buffer at pH 8.0 (see example 1). In another embodiment, aPn3Pase protein displays a concentration-dependent preference for Ca2+,as it produces a higher concentration of reducing end GlcA in presenceof 10 mM Ca2+(see example 1).

A Pn3Pase protein described herein can be expressed as a fusion proteinthat includes a Pn3Pase protein described herein and heterologous aminoacids. For instance, the additional amino acid sequence may be usefulfor purification of the fusion protein by affinity chromatography. Aminoacid sequences useful for purification can be referred to as a tag, andinclude but are not limited to a polyhistidine-tag (His-tag) andmaltose-binding protein. Representative examples may be found in Hopp etal. (U.S. Pat. No. 4,703,004), Hopp et al. (U.S. Pat. No. 4,782,137),Sgarlato (U.S. Pat. No. 5,935,824), and Sharma Sgarlato (U.S. Pat. No.5,594,115). Various methods are available for the addition of suchaffinity purification moieties to proteins. Optionally, the additionalamino acid sequence, such as a His-tag, can then be cleaved.

Polynucleotides

Also provided herein are isolated polynucleotides encoding a Pn3Paseprotein. A polynucleotide encoding a protein having Pn3Pase activity isreferred to herein as a Pn3Pase polynucleotide. Pn3Pase polynucleotidesmay have a nucleotide sequence encoding a protein having the amino acidsequence shown in SEQ ID NO:2, or a portion thereof. An example of theclass of nucleotide sequences encoding such a protein is SEQ ID NO:1 ora portion thereof. It should be understood that a polynucleotideencoding a Pn3Pase protein represented by SEQ ID NO:2 is not limited tothe nucleotide sequence disclosed at SEQ ID NO:1, but also includes theclass of polynucleotides encoding such proteins as a result of thedegeneracy of the genetic code. For example, the naturally occurringnucleotide sequence SEQ ID NO:1 is but one member of the class ofnucleotide sequences encoding a protein having the amino acid sequenceSEQ ID NO:2. The class of nucleotide sequences encoding a selectedprotein sequence is large but finite, and the nucleotide sequence ofeach member of the class may be readily determined by one skilled in theart by reference to the standard genetic code, wherein differentnucleotide triplets (codons) are known to encode the same amino acid.

A Pn3Pase polynucleotide may have sequence similarity with thenucleotide sequence of SEQ ID NO:1 or a portion thereof, for instancenucleotides encoding a mature Pn3Pase protein. Pn3Pase polynucleotideshaving sequence similarity with the nucleotide sequence of SEQ ID NO:1,or a portion thereof, encode a Pn3Pase protein. A Pn3Pase polynucleotidemay be isolated from a microbe, such as Paenibacillus sp., or may beproduced using recombinant techniques, or chemically or enzymaticallysynthesized. A Pn3Pase polynucleotide may further include heterologousnucleotides flanking the open reading frame encoding the Pn3Paseprotein. Typically, heterologous nucleotides may be at the 5′ end of thecoding region, at the 3′ end of the coding region, or the combinationthereof. The number of heterologous nucleotides may be, for instance, atleast 10, at least 100, or at least 1000.

A polynucleotide described herein may be present in a vector. A vectoris a replicating polynucleotide, such as a plasmid, phage, or cosmid, towhich another polynucleotide may be attached so as to bring about thereplication of the attached polynucleotide. Construction of vectorscontaining a polynucleotide of the disclosure employs standard ligationtechniques known in the art. See, e.g., Sambrook et al, MolecularCloning: A Laboratory Manual., Cold Spring Harbor Laboratory Press(1989). A vector may provide for further cloning (amplification of thepolynucleotide), i.e., a cloning vector, or for expression of thepolynucleotide, i.e., an expression vector. The term vector includes,but is not limited to, plasmid vectors, viral vectors, cosmid vectors,and artificial chromosome vectors. Examples of viral vectors include,for instance, adenoviral vectors, adeno-associated viral vectors,lentiviral vectors, retroviral vectors, and herpes virus vectors.Typically, a vector is capable of replication in a microbial host, forinstance, a microbe such as E. coli, or in a eukaryotic host, forinstance, a yeast cell, a mammalian cell, or an insect cell. In oneembodiment the vector is a plasmid.

Selection of a vector depends upon a variety of desired characteristicsin the resulting construct, such as a selection marker, vectorreplication rate, and the like. In some aspects, suitable host cells forcloning or expressing the vectors herein include prokaryotic cells.Vectors may be introduced into a host cell using methods that are knownand used routinely by the skilled person. For example, calcium phosphateprecipitation, electroporation, heat shock, lipofection, microinjection,and viral-mediated nucleic acid transfer are common methods forintroducing nucleic acids into host cells.

Polynucleotides encoding a Pn3Pase protein may be obtained frommicrobes, for instance, a microbe, such as Paenibacillus sp., orproduced in vitro or in vivo. For instance, methods for in vitrosynthesis include, but are not limited to, chemical synthesis with aconventional DNA/RNA synthesizer. Commercial suppliers of syntheticpolynucleotides and reagents for such synthesis are well known.

An expression vector optionally includes regulatory sequences operablylinked to the coding region. The disclosure is not limited by the use ofany particular promoter, and a wide variety of promoters are known.Promoters act as regulatory signals that bind RNA polymerase in a cellto initiate transcription of a downstream (3′ direction) coding region.The promoter used may be a constitutive or an inducible promoter. It maybe, but need not be, heterologous with respect to the host cell. Thepromoter useful in methods described herein may be, but is not limitedto, a constitutive promoter, a temperature sensitive promoter, anon-regulated promoter, or an inducible promoter. In one embodiment, apromoter is one that functions in a member of the domain Bacteria. Inone embodiment, a promoter is one that functions in a eukaryote.

An expression vector may optionally include a ribosome binding site anda start site (e.g., the codon ATG) to initiate translation of thetranscribed message to produce the protein. It may also include atermination sequence to end translation. A termination sequence istypically a codon for which there exists no correspondingaminoacetyl-tRNA, thus ending protein synthesis. The polynucleotide usedto transform the host cell may optionally further include atranscription termination sequence.

A vector introduced into a host cell to result in a genetically modifiedcell optionally includes one or more marker sequences, which typicallyencode a molecule that inactivates or otherwise detects or is detectedby a compound in the growth medium. For example, the inclusion of amarker sequence may render the transformed cell resistant to anantibiotic, or it may confer compound-specific metabolism on thetransformed cell. Examples of a marker sequence include, but are notlimited to, sequences that confer resistance to kanamycin, ampicillin,chloramphenicol, tetracycline, streptomycin, and neomycin.

Proteins described herein may be produced using recombinant DNAtechniques, such as an expression vector present in a cell. Such methodsare routine and known in the art. The proteins may also be synthesizedin vitro, e.g., by solid phase peptide synthetic methods. The solidphase peptide synthetic methods are routine and known in the art. Aprotein produced using recombinant techniques or by solid phase peptidesynthetic methods may be further purified by routine methods, such asfractionation on immunoaffinity or ion-exchange columns, ethanolprecipitation, reverse phase HPLC, chromatography on silica or on ananion-exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammoniumsulfate precipitation, gel filtration using, for example, Sephadex G-75,or ligand affinity.

Genetically Modified Cells

Also provided is a genetically modified cell having a polynucleotideencoding a Pn3Pase described herein. Compared to a control cell that isnot genetically modified, a genetically modified cell can exhibitproduction of a Pn3Pase, or can exhibit increased production of aPn3Pase. A polynucleotide encoding a Pn3Psae may be present in the cellas a vector or integrated into genomic DNA, such as a chromosome or aplasmid, of the genetically modified cell. A cell can be a eukaryoticcell or a prokaryotic cell, such as a member of the domain Bacteria

Examples of host cells that are members of the domain Bacteria that canbe genetically modified to include a polynucleotide encoding a Pn3Pasedescribed herein include, but are not limited to, Escherichia (such asEscherichia coli), and Salmonella (such as Salmonella enterica,Salmonella typhi, Salmonella typhimurium).

Examples of host cells that are eukaryotic cells that can be geneticallymodified to include a polynucleotide encoding a Pn3Pase include, but arenot limited to, yeast such as Saccharomyces cerevisiae and Pichia spp.,insect cells, and mammalian cells.

Compositions

Also provided are compositions that include a Pn3Pase protein describedherein or a polynucleotide encoding a Pn3Pase protein. Such compositionstypically include a pharmaceutically acceptable carrier. As used herein“pharmaceutically acceptable carrier” includes saline, solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. Additional active agents can also beincorporated into the compositions.

A composition may be prepared by methods well known in the art ofpharmaceutics. In general, a composition can be formulated to becompatible with its intended route of administration. Administration maybe systemic or local. Examples of routes of administration includeparenteral (e.g., intravenous, intradermal, subcutaneous,intraperitoneal, intramuscular), enteral (e.g., oral), and topical(e.g., epicutaneous, inhalational, transmucosal) administration.Appropriate dosage forms for enteral administration of the compound ofthe present disclosure include, but are not limited to, tablets,capsules or liquids. Appropriate dosage forms for parenteraladministration may include intravenous or intraperitonealadministration. Appropriate dosage forms for topical administrationinclude, but are not limited to, nasal sprays, metered dose inhalers,dry-powder inhalers or by nebulization.

Solutions or suspensions can include the following components: a sterilediluent such as water for administration, saline solution, fixed oils,polyethylene glycols, glycerin, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates; electrolytes, such as sodium ion,chloride ion, potassium ion, calcium ion, and magnesium ion, and agentsfor the adjustment of tonicity such as sodium chloride or dextrose. pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide.

Compositions can include sterile aqueous solutions (where water soluble)or dispersions and sterile powders for the extemporaneous preparation ofsterile solutions or dispersions. For parenteral administration,suitable carriers include physiological saline, bacteriostatic water,phosphate buffered saline (PBS), and the like. A composition istypically sterile and, when suitable for injectable use, should be fluidto the extent that easy syringability exists. It should be stable underthe conditions of manufacture and storage and preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. Prevention of the action of microorganisms can be achieved byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile solutions can be prepared by incorporating the active compound(e.g., a Pn3Pase protein described herein or a polynucleotide encodingthe protein) in the required amount in an appropriate solvent with oneor a combination of ingredients routinely used in pharmaceuticalcompositions, as required, followed by filtered sterilization.Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle, which contains a basic dispersion medium and anyother appropriate ingredients. In the case of sterile powders for thepreparation of sterile injectable solutions, preferred methods ofpreparation include vacuum drying and freeze-drying which yields apowder of the active ingredient plus any additional desired ingredientfrom a previously sterilized solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier. Pharmaceutically compatiblebinding agents and/or other useful materials can be included as part ofthe composition. The tablets, pills, capsules, troches and the like cancontain any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a disintegrating agentsuch as alginic acid, Primogel, or corn starch; a lubricant such asmagnesium stearate or Sterotes; a glidant such as colloidal silicondioxide; a sweetening agent such as sucrose or saccharin; or a flavoringagent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation (e.g., topical administration), theactive compounds can be delivered in the form of an aerosol spray from apressured container or dispenser which contains a suitable propellant,e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

In those embodiments where a polynucleotide encoding a recombinantprotein is administered, any method suitable for administration ofpolynucleotide agents can be used, such as gene guns, bio injectors, andskin patches as well as needle-free methods such as micro-particle DNAvaccine technologies (Johnston et al., U.S. Pat. No. 6,194,389).

The active compounds may be prepared with carriers that will protect thecompound against rapid elimination from the body, such as a controlledrelease formulation, including implants. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Suchformulations can be prepared using standard techniques. Liposomalsuspensions can also be used as pharmaceutically acceptable carriers.These can be prepared according to methods known to those skilled in theart.

Toxicity and therapeutic efficacy of the active compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD50 (the dose lethal to50% of the population) and the ED50 (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD50/ED50. Recombinant proteins exhibiting high therapeutic indices arepreferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration used. For a compound used inthe methods described herein, the therapeutically effective dose can beestimated initially from animal models. A dose may be formulated inanimal models to achieve a circulating plasma concentration range thatincludes the IC50 (i.e., the concentration of the test compound whichachieves a half-maximal inhibition of signs of disease, such asobesity). Such information can be used to more accurately determineuseful doses in humans. Levels in plasma may be measured using routinemethods.

The compositions can be administered one or more times per day to one ormore times per week, including once every other day. The skilled artisanwill appreciate that certain factors may influence the dosage and timingrequired to effectively treat a subject, including but not limited tothe severity of the condition, previous treatments, the general healthand/or age of the subject, and other diseases present. Moreover,treatment of a subject with an effective amount of an active compoundcan include a single treatment or, preferably, can include a series oftreatments.

Methods of Use

Also provided are methods. In one embodiment, a method is for makingPn3Pase protein described herein. In one embodiment, the method includesincubating a cell under suitable conditions for expression of a Pn3Paseprotein. The cell can be, but it not limited to, a genetically modifiedcell or a naturally occurring cell that produces a Pn3Pase protein. Anexample of a naturally occurring cell is a Paenibacillus strain, such asPaenibacillus sp. 32352. Optionally, the method includes introducinginto a host cell a vector that includes a coding region encoding aPn3Pase protein. In one embodiment, the method includes isolating orpurifying the Pn3Pase protein from a cell or from a medium. In thoseembodiments where the Pn3Pase protein includes additional amino acidsuseful for isolating or purifying the protein, the method can alsoinclude cleavage of the additional amino acids from the Pn3Pase protein.

In one embodiment, a method is for cleaving a Pn3P molecule. In oneembodiment, the method includes exposing a Pn3P molecule to a Pn3Paseprotein. The P3nP molecule can be part of a Streptococcus pneumoniaemicrobe, e.g., the Pn3P molecule can be the capsular polysaccharide ofan S. pneumoniae, or the Pn3P molecule can be separate from an S.pneumoniae microbe, e.g., the Pn3P molecule can be isolated. In oneembodiment, the S. pneumoniae microbe is serotype 3. In one embodiment,the Pn3P molecule is in vivo, and in another embodiment, the Pn3Pmolecule is in vitro.

In one embodiment, a method is for reducing the amount of type IIIcapsular polysaccharide on the surface of Streptococcus pneumoniae. Themethod includes contacting a Streptococcus pneumoniae having type IIIcapsular polysaccharide present on its surface with a Pn3Pase protein.The Pn3Pase protein can be isolated or purified, and can be present in acomposition. In one embodiment, the contacting can include exposing themicrobe to a genetically modified cell that expresses the Pn3Paseprotein. The contacting can be under conditions suitable for enzymatichydrolysis of type III capsular polysaccharide. Optionally, the theStreptococcus pneumoniae with the reduced amount of type III capsularpolysaccharide has increased susceptibility to phagocytosis bymacrophages, increased complement-mediated killing by neutrophils, or acombination thereof, compared to the Streptococcus pneumoniae that isnot contacted with the Pn3Pase protein. In one embodiment, the S.pneumoniae microbe is serotype 3. In one embodiment, the S. pneumoniaemicrobe is in vivo, and in another embodiment, the S. pneumoniae microbeis in vitro.

In one embodiment, a method is for increasing deposition of at least onecomplement component on the surface of Streptococcus pneumoniae. Themethod includes contacting a Streptococcus pneumoniae having type IIIcapsular polysaccharide present on its surface with a Pn3Pase protein.The Pn3Pase protein can be isolated or purified, and can be present in acomposition. In one embodiment, the contacting can include exposing themicrobe to a genetically modified cell that expresses the Pn3Paseprotein. The contacting can be under conditions suitable for enzymatichydrolysis of type III capsular polysaccharide. In one embodiment, theS. pneumoniae microbe is serotype 3. In one embodiment, the S.pneumoniae microbe is in vivo, and in another embodiment, the S.pneumoniae microbe is in vitro.

In one embodiment, a method includes treating an infection in a subjectcaused by Streptococcus pneumoniae. The subject used in a methoddescribed herein can be an animal such as, but not limited to, a murine(e.g., mouse or rat) or a human. The method includes administering aneffective amount of the composition to an animal having an infectioncaused by Streptococcus pneumoniae. Optionally, the method can includedetermining whether the Streptococcus pneumoniae causing the infectionhas decreased. Methods for determining whether an infection is caused bya Streptococcus pneumoniae are routine and known in the art. Theinfection can be localized or systemic. An example of a localizedStreptococcus pneumoniae infection is colonization of the nasal cavity,e.g., the nasopharynx. In one embodiment, topical administration of acomposition described herein can be used to reduce nasopharyngealcolonization in a subject. Another example of a localized Streptococcuspneumoniae infection of the lung, e.g., pneumococcal pneumonia. In oneembodiment, topical administration of a composition by, for instance,aerosol, can be used to reduce pneumococcal pneumonia in a subject. Anexample of a systemic Streptococcus pneumoniae infection is the presenceof Streptococcus pneumoniae in the blood of a subject (e.g., bacteremiaor sepsis). In one embodiment, parenteral administration of acomposition can be used to reduce a systemic Streptococcus pneumoniaeinfection in a subject. In this aspect of the disclosure, an “effectiveamount” of a composition of the present disclosure is the amount able toelicit the desired response in the recipient, e.g., a reduction in theamount of Streptococcus pneumoniae present in a subject. The reductioncan be a reduction in the number of S. pneumoniae in the nasopharynx,lung, or blood of a subject. The reduction can be a decrease of at least2-fold, at least 3-fold, or at least 4-fold in the subject compared tothe subject before administering the composition. In one embodiment, theS. pneumoniae microbe is serotype 3.

In another embodiment, a method includes treating one or more symptomsof certain conditions in animals that may be caused by infection by aStreptococcus pneumoniae. Streptococcus pneumoniae infections causeinvasive pneumococcal disease. Examples of conditions caused by invasivepneumococcal disease include pneumonia, pneumococcal meningitis, otitismedia, bacteremia, and sepsis. Symptoms associated with these conditionsinclude chills, cough, rapid breathing, difficulty breathing, chest pain(pneumonia), stiff neck, fever, headache, confusion and photophobia(pneumococcal meningitis), and confusion, shortness of breath, elevatedheart rate, pain or discomfort, over-perspiration, fever, and shivering(sepsis). In one embodiment, the S. pneumoniae microbe is serotype 3.

Treatment of one or more of these conditions can be prophylactic or,alternatively, can be initiated after the development of a conditiondescribed herein. Treatment that is prophylactic, for instance,initiated before a subject manifests a symptom of a condition caused byStreptococcus pneumoniae, is referred to herein as treatment of asubject that is “at risk” of developing the condition. Typically, ananimal “at risk” of developing a condition is an animal likely to beexposed to a Streptococcus pneumoniae causing the condition.Accordingly, administration of a composition can be performed before,during, or after the occurrence of the conditions described herein.Treatment initiated after the development of a condition may result indecreasing the severity of the symptoms of one of the conditions,including completely removing the symptoms. In this aspect of thedisclosure, an “effective amount” is an amount effective to prevent themanifestation of symptoms of a condition, decrease the severity of thesymptoms of a condition, and/or completely remove the symptoms. In oneembodiment, the S. pneumoniae microbe is serotype 3.

The potency of a composition described herein can be tested according tostandard methods. For instance, the use of mice as an experimental modelfor Streptococcus pneumoniae infection in humans is well established.

Kits

The present disclosure also provides a kit for making a Pn3Pase protein.In one embodiment, the kit includes a vector that includes a codingregion encoding a Pn3Pase protein in an amount sufficient fortransforming a cell. In one embodiment, the kit includes a geneticallymodified cell that includes a coding region encoding a Pn3Pase proteinin a suitable packaging material.

In another embodiment, the present disclosure also provides a kitdirected to using a Pn3Pase protein. In one embodiment, the kit includesPn3Pase protein, isolated or optionally purified, in a suitablepackaging material.

Optionally, other reagents such as buffers or a pharmaceuticallyacceptable carrier (either prepared or present in its constituentcomponents, where one or more of the components may be premixed or allof the components may be separate), and the like, are also included. Inone embodiment, the protein, vector, or genetically modified cell may bepresent with a buffer, or may be present in separate containers.Instructions for use of the packaged components are also typicallyincluded.

As used herein, the phrase “packaging material” refers to one or morephysical structures used to house the contents of the kit. The packagingmaterial is constructed by known methods, preferably to provide asterile, contaminant-free environment. The packaging material has alabel, which indicates that the contents can be used for transforming acell or producing Pn3Pase protein. In addition, the packaging materialcontains instructions indicating how the materials within the kit areused. As used herein, the term “package” refers to a solid matrix ormaterial such as glass, plastic, paper, foil, and the like, capable ofholding within fixed limits a vector or a genetically modified cell.Thus, for example, a package can include a glass or plastic vial used tocontain appropriate quantities of a Pn3Pase protein. “Instructions foruse” typically include a tangible expression describing the reagentconcentration or at least one method parameter.

ILLUSTRATIVE ASPECTS

Aspect 1. A genetically modified cell comprising a mature Pn3Paseprotein, wherein the protein has Pn3Pase activity.

Aspect 2. A genetically modified cell comprising a polynucleotidecomprising a coding region, wherein the coding region comprises anucleotide sequence encoding a mature Pn3Pase protein, and wherein theprotein has Pn3Pase activity.

Aspect 3. The genetically modified cell of Aspect 1 or 2 wherein theprotein comprises an amino acid sequence having at least 80% identitywith an amino acid sequence selected from SEQ ID NO:2, wherein the aminoterminal amino acid is selected from any one of residues 2 to 64 of SEQID NO:2 and the carboxy terminal amino acid is residue 1545 of SEQ IDNO:2.

Aspect 4. The genetically modified cell of any one of Aspects 1-3wherein the protein comprises an amino acid sequence having at least 80%identity with amino acids 41-1545 of SEQ ID NO:2.

Aspect 5. The genetically modified cell of any one of Aspects 1-4wherein the cell is a eukaryotic cell.

Aspect 6. The genetically modified cell of any one of Aspects 1-5wherein the cell is a mammalian cell, a yeast cell, or an insect cell.

Aspect 7. The genetically modified cell of any one of Aspects 1-6wherein the cell is a prokaryotic cell.

Aspect 8. The genetically modified cell of any one of Aspects 1-7wherein the cell is E. coli.

Aspect 9. The genetically modified cell of any one of Aspects 1-8wherein the protein comprises a heterologous amino acid sequence.

Aspect 10. The genetically modified cell of any one of Aspects 1-9wherein the heterologous amino acid sequence comprises a tag.

Aspect 11. A composition comprising the genetically modified cell of anyone of Aspects 1-10.

Aspect 12. A composition comprising an isolated mature P3nPase protein,wherein the protein has Pn3Pase activity.

Aspect 13. The composition of any one of Aspects 1-12 wherein theprotein is purified.

Aspect 14. A composition comprising an isolated polynucleotide, whereinthe polynucleotide comprises a coding region encoding a mature Pn3Paseprotein, and wherein the protein has Pn3Pase activity.

Aspect 15. The composition of any one of Aspects 12-14 wherein theprotein comprises an amino acid sequence having at least 80% identitywith an amino acid sequence selected from SEQ ID NO:2, wherein the aminoterminal amino acid is selected from residues 2 to 64 of SEQ ID NO:2 andthe carboxy terminal amino acid is residue 1545 of SEQ ID NO:2.

Aspect 16. The composition of any one of Aspects 12-15 wherein theprotein comprises an amino acid sequence having at least 80% identitywith amino acids 41-1545 of SEQ ID NO:2.

Aspect 17. The composition of any one of Aspects 11-16 wherein thecomposition comprises a pharmaceutically acceptable carrier.

Aspect 18. A method comprising: incubating a cell under conditionssuitable for expression of a protein having Pn3Pase activity, whereinthe cell comprises a polynucleotide comprising a coding region, whereinthe coding region comprises a nucleotide sequence encoding a maturePn3Pase protein, wherein the protein has Pn3Pase activity, and whereinthe cell expresses the mature Pn3Pase protein.

Aspect 19. The method of Aspect 18 wherein the protein comprises anamino acid sequence having at least 80% identity with an amino acidsequence selected from SEQ ID NO:2, wherein the amino terminal aminoacid is selected from any one of residues 2 to 64 of SEQ ID NO:2 and thecarboxy terminal amino acid is residue 1545 of SEQ ID NO:2.

Aspect 20. The method of any one of Aspects 18-19 wherein the proteincomprises an amino acid sequence having at least 80% identity with aminoacids 41-1545 of SEQ ID NO:2.

Aspect 21. The method of any one of Aspects 18-20 wherein the cell is agenetically modified cell and the polynucleotide is an exogenouspolynucleotide.

Aspect 22. The method of any one of Aspects 18-21 further comprisingisolating the protein.

Aspect 23. The method of any one of Aspects 18-21 further comprisingpurifying the protein.

Aspect 24. The method of any one of Aspects 18-21 wherein the cell is aeukaryotic cell.

Aspect 25. The method of any one of Aspects 18-24 wherein the cell is amammalian cell, a yeast cell, or an insect cell.

Aspect 26. The method of any one of Aspects 18-25 wherein the cell is aprokaryotic cell.

Aspect 27. The method of any one of Aspects 18-26 wherein theprokaryotic cell is E. coli.

Aspect 28. The method of any one of Aspects 18-27 wherein the proteincomprises a heterologous amino acid sequence.

Aspect 29. The method of any one of Aspects 18-28 wherein theheterologous amino acid sequence comprises a tag.

Aspect 30. A method comprising:

-   -   contacting a Streptococcus pneumoniae comprising a type III        capsular polysaccharide with a mature Pn3Pase protein comprising        Pn3Pase activity, wherein the contacting is under conditions        suitable for enzymatic hydrolysis of type III capsular        polysaccharide, wherein the amount of type III capsular        polysaccharide on the surface of the S. pneumoniae is reduced        compared to the S. pneumoniae that is not contacted with the        Pn3Pase protein.

Aspect 31. A method for increasing deposition of at least one complementcomponent on the surface of a Streptococcus pneumoniae, the methodcomprising:

-   -   contacting a Streptococcus pneumoniae comprising a type III        capsular polysaccharide with a mature Pn3Pase protein comprising        Pn3Pase activity, wherein the deposition of at least one        complement component on the surface of the S. pneumoniae is        increased compared to the S. pneumoniae that is not contacted        with the Pn3Pase protein.

Aspect 32. The method of Aspect 30 or 31 wherein the protein comprisesan amino acid sequence having at least 80% identity with an amino acidsequence selected from SEQ ID NO:2, wherein the amino terminal aminoacid is selected from any one of residues 2 to 64 of SEQ ID NO:2 and thecarboxy terminal amino acid is residue 1545 of SEQ ID NO:2.

Aspect 33. The method of any one of Aspects 30-32 wherein the proteincomprises an amino acid sequence having at least 80% identity with aminoacids 41-1545 of SEQ ID NO:2.

Aspect 34. The method of any one of Aspects 30-33 wherein the S.pneumoniae is present in conditions suitable for replication of the S.pneumoniae.

Aspect 35. The method of any one of Aspects 30-34 wherein the maturePn3Pase protein is an isolated Pn3Pase protein.

Aspect 36. The method of any one of Aspects 30-35 wherein the contactingcomprises exposing the S. pneumoniae to a genetically modified cell thatexpresses the mature Pn3Pase protein.

Aspect 37. The method of any one of Aspects 30-36 wherein the S.pneumoniae has increased susceptibility to phagocytosis by macrophages,increased complement-mediated killing by neutrophils, or a combinationthereof, compared to the S. pneumoniae that is not contacted with thePn3Pase protein.

Aspect 38. The method of any one of Aspects 30-37 wherein the S.pneumoniae is present in a subject.

Aspect 39. A method for treating an infection in a subject, the methodcomprising: administering an effective amount of a compositioncomprising a mature Pn3Pase protein having Pn3Pase activity to a subjecthaving or at risk of having an infection caused by a serotype 3 S.pneumoniae.

Aspect 40. A method for treating a symptom in a subject, the methodcomprising:

-   -   administering an effective amount of a composition comprising a        mature Pn3Pase protein having Pn3Pase activity to a subject        having or at risk of having an infection caused by a serotype        3 S. pneumoniae.

Aspect 41. A method for decreasing colonization in a subject, the methodcomprising:

-   -   administering an effective amount of a composition comprising a        comprising a mature Pn3Pase protein having P3nPase activity to a        subject colonized by or at risk of being colonized by a serotype        3 S. pneumoniae.

Aspect 42. The method of any one of Aspects 39-41 wherein the proteincomprises an amino acid sequence having at least 80% identity with anamino acid sequence selected from SEQ ID NO:2, wherein the aminoterminal amino acid is selected from any one of residues 2 to 64 of SEQID NO:2 and the carboxy terminal amino acid is residue 1545 of SEQ IDNO:2.

Aspect 43. The method of any one of Aspects 39-42 wherein the proteincomprises an amino acid sequence having at least 80% identity with aminoacids 41-1545 of SEQ ID NO:2.

Aspect 44. The method of any one of Aspects 39-43 wherein the subject isa human.

Aspect 45. A mature Pn3Pase protein disclosed herein for use in therapy.

Aspect 46. A mature Pn3Pase protein disclosed herein for use as amedicament.

Aspect 47. A mature Pn3Pase protein disclosed herein for use in thetreatment of a condition.

Aspect 48. Use of a mature Pn3Pase protein disclosed herein forpreparation of a medicament for the treatment of pneumonia, pneumococcalmeningitis, otitis media, bacteremia, sepsis, or a combination thereof.

Aspect 49. The composition comprising a mature P3nPase protein describedherein for use in treating or preventing an infection or a symptomcaused by a serotype 3 S. pneumoniae.

Aspect 50. A protein, composition, or method including one or morefeatures described herein.

EXAMPLES

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

Example 1 Identification of the Streptococcus pneumoniae Type 3Capsule-Specific Glycosyl Hydrolase Gene of Paenibacillus sp. 32352Abstract

‘Bacillus circulans Jordan 32352’ was isolated from decaying organicmatter in the New Jersey soil in the early 1930s. This soil-dwellingbacterium produced an enzyme capable of degrading the type 3 capsularpolysaccharide (Pn3P) of Streptococcus pneumoniae (Spn). Early reportsof this enzyme (Pn3Pase) demonstrated its inducibility by, andspecificity for Pn3P. A number of studies since have employed Pn3Pasewhile investigating Pn3P biosynthesis and its antigenic properties. Weset out to clone this enzyme for recombinant expression. We firstsequenced the genome of this bacterial species and reclassified thePn3Pase producing bacterium as “Paenibacillus sp. 32352”. Here, weidentified the gene of Pn3Pase through mass spectrometry-basedproteomics. We cloned the gene for recombinant expression andcharacterized the oligosaccharide products generated upon the enzymaticdepolymerization of Pn3P. We examined the effects of Pn3Pase on live,growing type 3 Spn, so that it may be investigated as a potentialtherapeutic agent against this hypervirulent serotype of Spn.

In 1930, Avery and Dubos isolated an organism from soil taken fromcranberry bogs, which was capable of depolymerizing type 3 capsularpolysaccharide of Streptococcus pneumoniae (Pn3P), a linear polymer of−3)βGlcA(1-4)βGlc(1-(1,2). The expression of this enzyme (Pn3Pase) wasinducible in the presence of Pn3P, and the bacterium was able to growwith Pn3P as the sole carbon source. They described this bacterium as asporulating, gram-negative, aerobic bacillus with peritrichous flagella.A few years later, Sickles and Shaw isolated two additional similarstrains demonstrating the same enzymatic activity targeting Pn3P (3,4).These strains were designated as Bacillus palustris, before beingaccepted as synonymous to Bacillus circulans (4-6). Besides Pn3Pase,these strains demonstrated the ability to produce enzymes capable ofdepolymerizing S. pneumoniae capsular serotypes 2 and 8, althoughsoluble protein in cell free extracts were inactive against thesepolysaccharides (3,7). There are numerous possible practicalapplications for these strains and enzymes. For example, investigatorshave applied the Sickles and Shaw Pn3Pase enzyme while examining Pn3Pbiosynthesis as well as its antigenic and immunological properties(8-11).

We obtained the ‘Bacillus Circulans Jordan strain 32352’ (i.e., theSickles and Shaw strain) from American Type Culture Collection, andsequenced its genome (12). 16S rRNA analysis revealed that thisbacterium belonged in the Paenibacillae genus, and it is now identifiedas Paenibacillus sp. 32352 (Pbac). Paenibacillus species are of growinginterest since the genus was established in 1991 (13). These microbesare a rich source of extracellular enzymes that catalyze a variety ofreactions, which have demonstrated utility in numerous agricultural andmedical applications (14-19). Database for carbohydrate-active enzymeannotation (dbCAN) of the Paenibacillus sp. 32352 genome indicates 665carbohydrate active entries out of 7200 predicted genes, 252 of thoseexhibiting glycosyl hydrolase- or polysaccharide lyase-like architecture(20).

We set out to determine the identity of the gene producing Pn3Pase inorder to express and utilize the enzyme's unique Pn3P-specific activity.We postulated the potential use of this enzyme as a therapeutic agentfor serotype 3 S. pneumoniae infections. The capsular polysaccharide(CPS) is a major virulence factor for type 3 strains, asnon-encapsulated mutants fail to colonize (21). The virulence mechanismsof CPS is to help Spn evade the immune system through resisting orinhibiting its phagocytosis by host macrophages while also limitingmucus-mediated clearance (22). The Pn3P component of the current13-valent vaccine (PCV13) induces variable immune responses to serotype3 (23,24). Individuals vaccinated with PCV13 require higheropsonophagocytosis assay serum titers for serotype 3 in comparison withother serotypes (25). Despite current vaccination programs against Spn,it remains one of the world's most lethal pathogens. In addition tovaccination and antibiotic delivery, alternative therapeutic approachesmust be considered for fighting these and other encapsulated pathogens.

Described here is the identification of the Paenibacillus Pn3Pasethrough proteomics of culture supernatant preparations with Pn3Psupplemented minimal media growth. We have cloned the Pn3Pase gene andexpressed active enzyme in E. coli. We have characterizedoligosaccharide products, and optimized conditions for Pn3Pdepolymerization. We assessed the ability of the enzyme to degrade thecapsule on a live virulent type 3 Spn strain to begin investigatingPn3Pase as a potential therapeutic agent.

Results

Pn3P Utilization Induces Expression of Genes Organized into Locus

We began by culturing Pbac in minimal M9 media with either 2% glucose orPn3P as the sole carbon source. Pbac growth was monitored for 14 h by OD600 nm to achieve maximum Pn3Pase production, (FIG. 3A). Pbac was ableto grow and utilize Pn3P and glucose, but was unable to utilizecellulose. Supernatants from these cultures were concentrated 20×, andproteins were visualized by coomassie staining. In the Pn3P growthconditions a prominent band ˜55 kDa was observed (FIG. 3B). Proteomicanalysis of these samples by in-solution trypsin digestion and LC-MSidentified numerous proteins that were present in both samples. Twoproteins, however, were significantly enriched in the Pn3P growthcondition as highlighted in Table 1.

TABLE 1 Proteomic identification of culture supernatant proteins ofPaenibacillus sp. 32352 grown in Pn3P. Highlighted are proteins uniqueto Pn3P grown culture. Gene Description Score Coverage # Unique Peptides# PSMs # AAs MW [kDa] Pbac_3554 *Lipoprotein 1301.24 82.02 49 597 50655.9 Pbac_1521 Ig-like, group 2, Surface layer protein 450.46 50.17 44218 1190 128.7 Pbac_6539 Hypothetical protein 237.19 31.20 17 82 57764.1 Pbac_5871 NLP/P60 family protein 103.53 61.54 6 35 156 16.9Pbac_1659 Alpha/beta hydrolase fold (EC 3.8.1.5) 94.98 66.07 13 72 28030.1 Pbac_3551 Hypothetical protein 39.14 16.70 6 53 1545 168.0 NCBI refseq: WP_079915027

These two Pn3P induced Pbac proteins appear to be organized into a locusof Pn3P utilization (FIG. 3C) consisting of ABC-type polysaccharidetransport system, permease component (Pbac_3556), probable ABCtransporter permease protein ytcP (Pbac_3555), lipoprotein (Pbac_3554),DNA-binding response regulator, AraC family (Pbac_3553), multidomainprotein with s-layer homology region, glug motif, ig motif (Pbac_3552),and hypothetical protein (Pbac_3551). Since two of these proteins aremore abundant in the Pn3P growth conditions, we compared thetranscription of three genes of this locus with the commonly expressedsurface layer protein (Pbac_1521) in the glucose and Pn3P samples byRT-PCR. The transcription of genes 3551, 3552, and 3554 increase˜130-fold with Pn3P utilization, while mRNA expression of Pbac_1521 isunchanged between the two conditions (FIG. 3D).

Pn3Pase Identification and Domain Analysis

Pbac_3554, Pbac_3552, and Pbac_3551 were cloned and expressed in E. coliBL21 (DE3) cells to determine which upregulated gene product wasresponsible for Pn3P depolymerase activity. The hypothetical proteinPbac_3551 (Accession WP_079915027), with primary amino acid sequenceshown in FIG. 4A, demonstrated rapid and efficient hydrolysis of tritiumradio-isotope labeled Pn3P, as shown by counts per minute shift to lowermolecular weight oligosaccharides when reaction products were separatedby size exclusion chromatography (FIG. 4B). Reactions of recombinantPn3Pase with unlabeled Pn3P were performed, spotted on a PVDF membrane,and probed with a Pn3P monoclonal antibody. Reactivity to the monoclonalantibody was completely abolished after 4 h Pn3Pase treatment of Pn3P(FIG. 4C).

The translated protein sequence of Pbac_3551 is 1,545 amino acids.Predicted cleavage of the signal peptide by SignalP 4.1 server (26) fromresidues 1-40 would yield a mature protein of 164.1 kDa. Proteinsequence analysis and classification by InterPro online software (27)recognized homology to glycoside hydrolase, family 39 from amino acids180-353, galactose-binding domain-like from 621-765, a domain of unknownfunction DUF1080 from 781-950 with structural similarity to anendo-1,3-1,4-beta glucanase, and a concanavalin A-like lectin/glucanasedomain from 1,209-1,348 (FIG. 4D). Although segments of this enzymedisplay some homology to numerous carbohydrate active proteins, nooverall homology to known glycosyl hydrolase families across the lengthof the enzyme exist.

Characterization of Oligosaccharide Products

The oligosaccharide products of Pn3Pase hydrolysis were separated bysize exclusion chromatography. This hydrolysis yielded two major productpeaks eluting late in the Superdex peptide column corresponding to atetrasaccharide and hexasaccharide (FIGS. 5A-C). The identities of thesepeaks were confirmed as tetrasaccharide (FIG. 5B) and hexasaccharide(FIG. 5C) by electrospray ionization mass spectrometry. Massspectrometry data is summarized in Table 2.

TABLE 2 Different charge states of the tetrasaccharide andhexasaccharide detected by mass spectrometry. m/z observed Charge stateExperimental M Theoretical M Error (ppm) Tetrasaccharide 693.1699 1694.1777 694.1804 −3.85 346.0811 2 694.1779 694.1804 −3.66Hexasaccharide 1031.2563 1 1032.2641 1032.2653 −1.13 515.1256 21032.2669 1032.2653 1.51

The characterization of oligosaccharide products by NMR spectroscopy ispresented in FIG. 6 . All anomeric proton signals are assigned inrepresentative ¹H NMR spectra of tetra- and hexa-saccharides (FIG. 6 ).Anomeric proton signals of residue A, C and E at ˜4.70 ppm wereoverlapped with HOD peak (FIGS. 6A, 4D) but showed up in 2D HSQCspectrum (FIGS. 6B, E). The ³J_(HH) coupling constants of B-1 and D-1were 8.22 Hz, demonstrating β-linkages. The signals at 5.15(³J_(HH)=3.69) and 4.58 (³J_(HH)=8.05) ppm correspond to α- andβ-configuration of GlcA residue F, respectively. By combining HSQC andCOSY experiments (FIGS. 6C, F), we were able to identify that protonsignal of F-5 possessed a high chemical shift (˜4.05 ppm) suggestingthat GlcA residue (F) was at the reducing end of the carbohydrate chain.These data demonstrate that Pn3Pase cleaves the β(1-4) linkage betweenglucuronic acid and glucose in the polysaccharide chain.

Pn3Pase Activity Analysis

A time course experiment was performed using tritiated Pn3P and therecombinant Pn3Pase to understand whether this enzymatic degradationproceeds through endolytic or exolytic cleavage. Reaction products wereseparated by size exclusion chromatography after proceeding for thegiven time. Low molecular weight oligosaccharides are generated early onin the reaction (FIG. 7A). Both an increase in CPM for theoligosaccharide elution volume, and corresponding decrease in CPM forthe higher molecular weight polymer over time suggest an exolytic typecleavage (FIG. 7A) that preferentially generates tetrasaccharides andhexasaccharides (FIG. 5A). A gradual shift of the peak from left toright over time would be indicative of true random endolytic cleavage.

Two-hour reactions were performed in three different buffers at pH 6.0,7.2, and 8.0, detecting the concentration of reducing end glucuronicacid by the p-hydroxybenzoic acid hydrazide (PAHBAH) method (43) todetermine the optimum reaction conditions for Pn3Pase. Pn3Pase displaysslightly better activity in sodium phosphate buffer at pH 7.2 than inMES buffer pH 6.0, but performs significantly worse in Tris buffer at pH8.0 in Tris buffer pH 8.0 (FIG. 7B). Further optimization focusing onmetal-ion dependence was performed with Mg²⁺ and Ca²⁺. Pn3Pase displaysa concentration-dependent preference for Ca²⁺, as it produces a higherconcentration of reducing end GlcA in presence of 10 mM Ca²⁺ (FIG. 7C).

Pn3Pase Removes Capsule from Growing Type 3 Streptococcus pneumoniae

The type 3 WU2 strain was cultured for 10 h with 100 μg/ml of therecombinant enzyme added to the growth medium to begin assessing effectsof Pn3Pase treatment of growing type 3 S. pneumoniae. Pn3Pase treatmentof cells demonstrated no adverse growth or cytotoxic effects on thebacterial growth as indicated in FIG. 8A. Additionally, comparable CFUvalues were obtained at two-hour intervals (data not shown).

The bacterial cells grown in the presence of 2 μg/ml or 10 μg/ml Pn3Pasewere then examined by competition ELISA to determine if the enzyme ledto efficient capsule shedding of the growing cultures. After treatment,fixed whole cells, at different concentrations, were used to compete forPn3P specific antibodies binding to the Pn3P coated plate. The acapsularWU2 mutant strain (JD908) showed minimal inhibition at 5×10⁵ CFU/ml andno inhibition at 5×10⁴ CFU/mL concentration, due to its lack of capsule(FIG. 8B). Heat inactivated Pn3Pase treated cells demonstrated thehighest inhibition percent as cell surfaces should be fully decoratedwith CPS (FIG. 8B) With active Pn3Pase treatment, inhibition of antibodybinding begins to decrease in a Pn3Pase concentration dependent manner(FIG. 8B), suggesting that the enzyme is well-designed to strip thecapsule from live, growing type 3 Streptococcus pneumoniae.

A time course experiment was performed in which the bacterial cells weregrown to mid-log phase, suspended in PBS, and treated with 1 μg/mlPn3Pase for 0, 1, 2, or 4 hours. The Pn3Pase dose was lowered for thisexperiment given that the WU2 strain should not be actively growing andproducing a substantial amount of new CPS under these conditions. Theenzymatic activity of the Pn3Pase is also slightly better in PBS than inthe THY culture media (data not shown). At the given time point, thetreated cells were fixed and examined by competition ELISA as describedabove. The acapsular WU2 mutant strain (JD908), again, showed minimalinhibition at 5×10⁵ CFU/ml and no inhibition at 5×10⁴ CFU/mLconcentration, due to its lack of capsule (FIG. 8C). Untreated cellsexhibited the highest inhibition percent, indicative of a cell surfacefully decorated with CPS (FIG. 8C) With increasing Pn3Pase incubationtime, inhibition of antibody binding begins to decrease significantly(FIG. 8C), appearing essentially acapsular after 2 and 4 hour treatment.

The Pn3Pase treated cells were visualized by transmission electronmicroscopy and compared to both untreated and an acapsular mutantstrain. The untreated cells displayed a thick, complete CPS coat acrosstheir surface (FIG. 8D upper left). As expected, the acapsular mutanthad no CPS (FIG. 8D upper right). The capsule layer of the enzymetreated cells exhibited minimal thickness in most cases (FIG. 8D bottomleft), while some looked as though they were acapsular (FIG. 8D bottomright).

Discussion

The Paenibacillus genus, literal Latin translation, “almost Bacillus,”was ruled distinct from true Bacillus species when phylogenetic analysison 16S rRNA gene sequences was performed for a number of strainspreviously defined as Bacillus (6). Sequence analysis showed thatseveral bacterial strains sorted into this genus, required reassignment.Species belonging to this genus have been obtained from diverseecological niches (28) from aquatic (29) to desert environments (30),and from hot springs (31) to extreme cold regions (32). ManyPaenibacillus species are found in soil (3,33) and plant rootenvironments (34), however, a number are isolated from human samples aswell (35). Paenibacillus species are a rich source for a variety ofagricultural, biomedical, and industrial products (28). Extracellularenzymes demonstrating numerous activites (36) have applications inproduction of a variety of industrially significant materials (28). Anumber of these species are efficient nitrogen fixers that have beenapplied agriculturally to promote crop growth (37). In addition,protective action of novel antimicrobial peptides and compounds obtainedfrom Paenibacilli has been demonstrated (38).

While Paenibacillus sp. 32352 was isolated from a soil source (3), it isappropriate to question the evolutionary pressure to obtain enzymescapable of acting on a human pathogen Spn CPS. Based on earlier studies,this particular strain demonstrates the ability to degrade threedistinct pneumococcal CPSs (3,9). Whether these are the naturalsubstrates for the enzymes, or whether other soil dwelling microbes orplant matter possess similar glycan residues and linkages remains to beinvestigated.

However originally acquired, Paenibacillus sp. 32352 has adapted thePn3Pase described here to play an important functional role in itsmetabolism. Early reports by Torriani and Pappenheimer on this speciesindicated the ability to induce Pn3Pase activity in culture supernatantby addition of Pn3P in the growth medium (7). The “inducibility” was incontrast to the Avery and Dubose findings that Pn3Pase formation onlyoccurred in conditions where Pn3P is present as a sole carbon source(2).Here, we demonstrate that while Pn3P is not required for bacterialgrowth, it can serve as the sole nutrition source. Moreover, our dataindicate that presence of Pn3P in the Pbac culture medium induces theexpression of Pn3Pase.

Our attempts to purify multi-milligram quantities of active, nativePn3Pase from an induced culture of this strain were unsuccessful, thoughPn3P depolymerizing activity can be detected in these culturesupernatant preparations. In this study, we have identified and clonedthe Pn3Pase gene from Paenibacillus sp. 32352. We have fullycharacterized the oligosaccharide products, and we have shown thatPn3Pase can rapidly strip the CPS from live, growing Spn. Future studieswill assess this enzyme's utility as a treatment for serotype 3 Spninfections. Inefficiencies in current vaccination and antibioticadministration necessitate the discovery of alternative therapeuticapproaches for controlling these and other encapsulated pathogens.

Experimental Procedures Bacterial Strains and Growth Conditions

Paenibacillus sp. 32352 (ATCC 14175) was cultured aerobically withshaking at 37° C. on Tryptic Soy Agar with 5% sheep blood (HardyDiagnostics), or in minimal medium (M9 Teknoba) culture containing 1 mMMgSO₄, 1 mM biotin, 1 mM thiamin, and 2% glucose (Sigma Aldrich) or Pn3Ppowder (ATCC 172-X) as sole carbon source. Streptococcus pneumoniae type3 (WU2 strain) and acapsular derivative (JD908), generous gifts fromMoon Nahm (University of Alabama at Birmingham), were culturedaerobically without shaking at 37° C. on Tryptic Soy Agar with 5% sheepblood (TSAB), or in Todd Hewitt Broth plus 0.5% yeast extract (THY) (BDBiosciences).

Proteomics of Culture Supernatants

Paenibacillus sp. 32352 was cultured in 5 ml of minimal medium M9containing 2% (w/v) carbon source, as described above. Bacterialcultures were harvested at mid-log phase (OD600 nm 0.6). Culturesupernatants were passed through a 0.45 μm syringe filter andconcentrated to 1/20^(th) culture volume using a microsep advancecentrifugal device with 10K molecular weight cutoff (Pall). Proteinconcentration was determined by bicinchoninic acid assay. An in-solutiontrypsin digestion was performed as described previously (39). Briefly,20 μg protein from culture supernatant protein was reduced by incubationwith 10 mM DTT for 1 h at 56° C., followed by carboxyamidomethylationwith 55 mM iodoacetamide in the dark at room temperature for 45 min, andthen digested with 1 Dg of sequencing grade trypsin (Promega) in 40 mMammonium bicarbonate overnight at 37° C. trypsin digest was stopped byaddition of 1% trifluoroacetic acid and incubation on ice for 30minutes. The resulting peptides were cleaned up using C18 spin columns(G Biosciences), dried down, and reconstituted in 0.1% formic acid. Thepeptides were separated using a Thermo Scientific UltiMate 3000 systemon a 15 cm Acclaim™ PepMap™ RSLC C18 Column (2 μm particle size, 75 μmID) using a 180 min linear gradient consisting of 1-100% Solvent B (80%acetonitrile, 0.1% formic acid) over 130 min at a flow rate of 200nL/min. Separated peptides were directly eluted into a nanospray ionsource of an Orbitrap Fusion Tribrid mass spectrometer (Thermo FisherScientific). The stainless steel emitter spray voltage was set to 2200V, and the temperature of the ion transfer tube was set to 280° C. FullMS scans were acquired using Orbitrap detection from m z 200 to 2000 at60,000 resolution, and MS² scans following fragmentation bycollision-induced dissociation (38% collision energy) were acquired inthe ion trap for the most intense ions in “Top Speed” mode using ThermoXcalibur Instrument Setup 3.0. The raw spectra were searched against theRapid Annotation Server(40) (RAST) annotated genome database forPaenibacillus sp. 32352 using SEQUEST in Proteome Discoverer 1.4 (ThermoFisher Scientific) with full MS peptide tolerance of 10 ppm and MS2peptide tolerance of 0.3 Da. Constant modification of +57.021 Da(carbamidomethylation of cysteine residues), and dynamic modification of+15.995 Da (oxidation of methionine residues) were allowed in the searchparameters. Results were filtered at a 1% false discovery rate forpeptide assignments.

Gene Expression

Comparison of the levels of transcript expression from the Pn3P inducedlocus was performed by RT-PCR. Paenibacillus sp. 32352 was cultured in 5ml of minimal medium containing 2% (w/v) carbon source, as describedabove. Triplicate bacterial cultures were harvested at mid-log phase (OD600 nm 0.6), RNA was purified using E.N.Z.A. Bacterial RNA Kit, followedby TRIzol (Thermo Fisher Scientific) extraction of RNA fromcontaminating genomic DNA as described previously (41). RNA purity wasassessed with nanodrop, and 1 μg of RNA was used for reversetranscription reaction using iscript CDNA synthesis kit (BioRad).Quantitative real time-PCR was performed in a 96-well plate on a MyiQsystem (BioRad) with iQ SYBR green mastermix. Primers used in RT-PCR arelisted in Table 3. The reactions were carried out in 20 μl, consistingof 10 μl of SYBR Green mix, 20 ng of cDNA, and 1 μM primer mix. Thereaction conditions were 95° C. 180 s, followed by 45 cycles of 95° C.for 10 s, 55° C. for 20 s, and 72° C. for 30 s. The data were normalizedto 16S rRNA transcript levels, and changes in expression level werecalculated as fold change compared with cultures of minimal medium withglucose supplement.

TABLE 3 Oligonucleotides used. Oligonucleotide Sequence (5′ to 3′)Pbac_3551F-RT gcacccgtgaatctggaagc (SEQ ID NO: 10) Pbac_3551R-RTctccataggaataagcgagagatagg (SEQ ID NO: 11) Pbac_3552F-RTgagccgtcaggcaccatac (SEQ ID NO: 12) Pbac_3552R-RTctctaatgcaggctgcgg (SEQ ID NO: 13) Pbac_3554F-RTggctgcggcggcacta (SEQ ID NO: 14) Pbac_3554R-RTtcccaaaacatcccggatcg (SEQ ID NO: 15) Pbac_1521F-RTgatggcgtgaaagacgatac (SEQ ID NO: 16) Pbac_1521R-RTacagttttgtcgttaacgcc (SEQ ID NO: 17) Pbac_16sF-RTcatgagggaatcatgaaacacg (SEQ ID NO: 18) Pbac_16sR-RTgggctttcttctcaggtacc (SEQ ID NO: 19) Pbac_3551Fggggacaagtttgtacaaaaaagcaggcttcgaaggagatagaaccatggcacccgtgaatctggaagc(SEQ ID NO: 20) Pbac_3551Rggggaccactttgtacaagaaagctgggtgctccacgatcaccttattcgataacg (SEQ ID NO: 21)Pbac_3552Fggggacaagtttgtacaaaaaagcaggcttcgaaggagatagaaccatggagccgtcaggcaccatac(SEQ ID NO: 22) Pbac_3552Rggggaccactttgtacaagaaagctgggtgcatccctcctgcgatgtg (SEQ ID NO: 23)Pbac_3554Fggggacaagtttgtacaaaaaagcaggcttcgaaggagatagaaccatgggctgcggcggcacta(SEQ ID NO: 24) Pbac_3554Rggggaccactttgtacaagaaagctgggtgcttctttgcgctcttgttatactc (SEQ ID NO: 25)

Production of Recombinant Pn3Pase

The coding region of Pbac_3551 (ref seq WP_079915027), Pbac_3552 (refseq WP_079915028), and Pbac_3554 (ref seq WP_079915030.1) (minuspredicted signal peptide and stop codon) were amplified fromPaenibacillus sp. 32352 genomic DNA (DNeasy blood and tissue kit,Qiagen) using 2× platinum superfi mastermix (Thermo Fisher Scientific)with overhang containing B-sites to facilitate gateway cloning (42) viaBP reaction (Thermo Fisher Scientific) into pDONR221. Primers forcloning are listed in Table 3. After DH5a transformation and DNAsequence confirmation, an LR-clonase reaction was performed to insertthe gene into the pET-DEST42 (Thermo Fisher Scientific) destinationvector for the expression of a carboxy-terminal His₆-tagged fusionprotein in E. coli BL21(DE3) cells. BL21(DE3) cells transformed with thepET-DEST42-“Pn3Pase” plasmid were grown in LB medium supplemented with100 μg/ml ampicillin at 37° C., and cell density was monitored byabsorbance at 600 nm. Once the OD 600 nm reached 0.6, the cells weretransferred into 25° C., protein expression was induced by the additionof Isopropyl β-D-1-thiogalactopyranoside to a final concentration of 1mM and the cell culture was allowed to incubate for 8 h (until A₆₀₀reached ˜1.1). Cells were harvested by centrifugation. Cells were thenresuspended in phosphate-buffered saline (PBS, pH 7.2) with 1 mg/mllysozyme for 20 min at 30° C., probe sonicated for 2 min (four cycles of20 s on, 10 s off), clarified by centrifugation at 17,000×g for 1 h at4° C., and passed through a 0.45 μm syringe filter. Recombinant Pn3Pasewas purified by Ni²⁺-NTA resin at 4° C., eluted with 300 mM imidazoleand buffer exchanged into PBS pH 7.2. Protein concentration wasdetermined by bicinchoninic acid assay. Purity was assessed byvisualizing proteins on stain free tris-glycine gel (BioRad) using geldoc EZ imager (BioRad).

Enzyme Assays

Tritiated Pn3P assays—Recombinant enzyme activity against type 3capsular polysaccharide was assayed by incubation of 2 μg/ml recombinantprotein, or heat killed control, with 10 μg/ml ³H-Pn3P in PBS. Thereaction was stopped after 2 h by heating at 100° C. for 5 minutes.Reaction mixture was separated on a superdex peptide 10/300 GL column(GE) on an NGC discoverer FPLC system (BioRad). Fractions of 1 ml werecollected, and counts per minute in each fraction were counted in aTri-Carb 2910 TR liquid scintillation analyzer (Perkin Elmer). Timecourse experiments were analyzed by the same method.

Reducing end sugar assays—recombinant Pn3Pase hydrolysis activity wasdetermined by measuring the increase in reducing ends using thep-hydroxybenzoic acid hydrazide (PAHBAH) method (43). A reaction mixture(200 μl) containing 20 μg Pn3P, and 1 μg recombinant Pn3Pase in either50 mM MOPS-NaOH (pH 6.0), 50 mM sodium phosphate buffer (pH 7.2), or 20mM Tris-HCl (pH 8.0), was incubated at 37° C. for 1 h and then heated at100° C. for 5 min to stop. Reaction mixture (40 μl) was mixed with 120μl of 1% (w/v) PAHBAH-HCL solution, heated at 100° C. for 5 min.Absorbance at 405 nm was measured on a Biotek synergy H1 microplatereader in a clear bottom 96-well microplate. Concentrations of reducingsugars were calculated based on GlcA standard curves generated by thesame method in respective buffers. Metal dependence assays wereperformed similarly with addition of MgCl₂ or CaCl₂) in phosphate buffer(pH 7.2).

Oligosaccharide Analysis

Pn3P powder (2 mg) was incubated with 100 μg of Pn3Pase at 37° C. for 48h. The reaction was stopped by heating at 100° C. for 5 min, and loadedonto Superdex peptide 10/300 GL column (GE). Products were separated inphosphate buffered saline at a flow rate of 1 ml/min and monitored byrefractive index. Fractions (0.5 ml) were collected and oligosaccharidepeaks were purified, desalted into water on a packed fine P2 column(Biorad). Desalted oligos were lyophilized and subject to ESI-MS formass determination and NMR for structural and reducing endidentification.

NMR—Oligosaccharides were dissolved in 400 μL ²H₂O (99.9%,Sigma-Aldrich, St. Louis, MO) and lyophilized three-times to remove theexchangeable protons. The samples were re-dissolved in 400 μl 99.96%²H₂O and transferred to NMR microtubes. ¹H spectroscopy, ¹³Cspectroscopy, ¹H-¹H correlated spectroscopy (COSY), and ¹H-¹³Cheteronuclear single quantum coherence spectroscopy (HSQC) experimentswere all performed at 298 K on Bruker 600 or 800 MHz spectrometer withTopspin 2.1.6 software.

Pn3Pase Treatment of Type 3 S. pneumoniae

Fresh Streptococcus pneumoniae type 3 (WU2) and acapsular derivative(JD908) colonies on TSAB plates were inoculated to 0.1 OD in THY brothand cultured as described above. WU2 growth in presence of 100 μg/mlPn3Pase was monitored over the course of 10 h by measuring OD 600 nm.WU2 grown in presence of 2 μg/ml, 10 μg/ml, or 10 μ/ml heat inactivatedPn3Pase, along with acapsular were grown for 6 hours, serially diluted,and plated to determine colony forming units. Cultures were harvested bycentrifugation, washed in PBS, fixed in 2% paraformaldehyde for 20 minon ice, washed once more, and suspended in 1 ml PBS. For time courseexperiment, WU2 and acapsular strain were grown to mid-log phase (OD 600nm of 0.6), harvested by centrifugation, washed in PBS, and thensuspended in 1 ml PBS. 1 μg/ml Pn3Pase was added and incubated at 37° C.for the 1, 2, or 4 hours. Treated cells were serially diluted, andplated to determine colony forming units, fixed in 2% paraformaldehydefor 20 min on ice, washed once more, and suspended in 1 ml PBS.

Competition ELISA

ELISA plates (96 well, Nunc) were coated with 5 μg/ml Pn3P in 0.1 Mcarbonate buffer (pH 9.0) overnight at room temperature. Plates werewashed 4-times with PBS +0.1% Tween (PBS-T) 20 using a Biotek 405/LSmicroplate washer. After 1 h blocking at room temperature with 1% BSA inPBS, microplate wells were incubated for 2 h at room temperature withfixed, treated cells that were pre-incubated for 30 min with Pn3Pspecific anti-serum in PBS-T. Plates were washed, and then incubated for2 h at room temperature with 1:2000 dilution of goat anti-mouse IgG-AP(Southern Biotech #1030-04) in PBS-T. After washing, plates wereincubated for ˜30 min at 37° C. with 2 mg/ml phosphatase substrate(Sigma S0942) in 1 M Tris 0.3 mM MgCl₂. Absorbance at 405 nm wasmeasured on a Biotek synergy H1 microplate reader. Percent inhibition ofantibody binding was calculated by((Uninhibited_(OD405)−Inhibited_(OD405))/Uninhibited_(OD405))×100.

Electron Microscopy

Electron microscopy was performed by Georgia Electron Microscopy corefacility at the University of Georgia according to a modified method byHammerschmidt et al. (44). Treated cells were fixed in 2% glutaraldehydeand 0.15% ruthenium red in PBS buffer for 1 h on ice and then rinsed 2×with buffer containing 0.15% ruthenium red, 15 min per rinse. Cells werethen fixed in 1% osmium tetroxide in buffer containing 0.15% rutheniumred for 1 h at room temperature followed by two rinses in buffercontaining 0.15% ruthenium red, 15 min per rinse. Pellet was dehydratedin a graded ethanol series (30%, 50%, 75%, 95%, 100% and 100%) and twochanges in 100% acetone, 15 min each step. Pellet was then infiltratedwith 25% Spurr's resin and 75% acetone—2 h followed by sequentialinfiltration with 50% Spurr's resin and 50% acetone, 75% Spurr's resinand 25% acetone, 100% Spurr's resin and then polymerized in a 70° C.oven for 24 h. Samples were sectioned at 60 nm with a Diatome diamondknife and pick up on slot grids. Grids were post-stained on drops ofuranyl acetate and lead citrate, 5 min each and rinsed with H₂O 30 sbetween stains. Samples were scoped using a JEOL JEM 1011 TEM (JEOL USA,Peabody, MA) operated at 80 kV.

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Example 2 Enzymatic Hydrolysis of Pneumococcal Capsular PolysaccharideRenders the Bacterium Vulnerable to Host Defense Abstract

Despite a century of investigation, Streptococcus pneumoniae (Spn)remains a major human pathogen, causing a number of diseases such aspneumonia, meningitis, and otitis media. Like many encapsulatedpathogens, the capsular polysaccharide (CPS) of Spn is a criticalcomponent for colonization and virulence in mammalian hosts. This studyaimed to evaluate the protective role of a glycoside hydrolase, Pn3Pase,targeting the CPS of type 3 Spn, which is one of the most virulentserotypes. We have assessed the ability of Pn3Pase to degrade thecapsule on a live type 3 strain. Through in vitro assays we observedthat Pn3Pase treatment increases the bacterium's susceptibility tophagocytosis by macrophages and complement-mediated killing byneutrophils. We have demonstrated that in vivo Pn3Pase treatment reducesnasopharyngeal colonization, and protects mice from sepsis caused bytype 3 Spn. Due to the increasing shifts in serotype distribution, risein drug resistant strains, and poor immune responses to vaccine-includedserotypes, it is necessary to investigate approaches to combatpneumococcal infections. This study evaluates the interaction ofpneumococcal CPS with host at molecular, cellular and systemic levelsand offers an alternative therapeutic approach for diseases caused bySpn through enzymatic hydrolysis of the CPS.

Introduction

Streptococcus pneumoniae (Spn), the causal agent of pneumonia,meningitis, and otitis media, remains a major threat to human health.This bacterium can stably colonize the human nasopharynx as a part ofthe normal commensal microflora (1-3). Colonization is the primary modeof transmission and a key step in the initiation of disease, despiteasymptomatic carriage (4, 5). A critical component for survival withinthe host and full pathogenicity of most Spn strains is the capsularpolysaccharide (CPS) (6, 7). The CPS is a large and distinctpolysaccharide structure coating the entire surface of the bacterium.The capsule helps Spn evade the host immune system through resisting orinhibiting its phagocytosis by host macrophages while also limitingmucus-mediated clearance (8-11). Spn has over 90 unique capsularserotypes, each differing in monosaccharide composition and linkage, aswell as other modifications such as acetylation (12). The requirement ofthe CPS in bacterial virulence, surface accessibility, and antigenicity,has made it a target in vaccination studies for over 100 years (12-16).Great strides have been made at increasing the immunogenicity andefficacy of pneumococcal vaccines that utilize the CPS by conjugation toa protein carrier (17, 18). Current pneumococcal vaccines aim to provideserotype-specific protection for some of the most relevant clinicalserotypes (12, 13). The use of the 7 and 13-valent pneumococcalconjugate vaccines, PCV7 and PCV13 (Prevnar; Pfizer) has been a majorsuccess; reducing invasive pneumococcal disease (IPD) ratessignificantly in both vaccinated and unvaccinated populations (15,19-22).

While the conjugate vaccines have been effective for preventing carriageand IPD caused by most included serotypes, the exception has beenserotype 3. The pneumococcal type 3 polysaccharide (Pn3P) component ofthe current PCV13 induces variable immune responses to Spn serotype 3,(23-25). Pn3P is a linear polymer of −3)βGlcA(1-4)βGlc(1-disacchariderepeating units with an average molecular weight of >400 kDa (26, 27).It was noted that significantly higher serum titers are required forserotype 3 in comparison with other serotypes for opsonophagocytickilling of Spn (12, 28, 29). Numerous animal model and epidemiologystudies have associated type 3 strains with increased virulence anddeath risk compared to other pneumococcal serotypes (30-32). A recentcase report demonstrated a fatal case of IPD caused by serotype 3,highlighting increased complications and generally poor outcomesassociated with this invasive serotype (33). In addition, recent dataindicate that Spn strains are resistant to one or more antibiotics in30% of IPD cases (34). The Centers for Disease Control and Preventionpredict a rise in antibiotic-resistance features of Spn (35, 36).

The inability of vaccination to provide adequate protection against oneof the most aggressive serotypes of this major human pathogennecessitates the urgent exploration of alternative approaches for type 3pneumococcal infections. This, along with a rise in the prevalence ofantibiotic resistant strains (37, 38) led us to revisit early studies byAvery and Dubos, who discovered a soil dwelling bacterium producing anenzyme that hydrolyzes Pn3P (39-41). Previously, we have identified thisbacterium as a Paenibacillus species, cloned its type 3 specificglycosyl hydrolase, Pn3Pase, and characterized its degradation products(26, 42). In light of the continued prevalence, and severity of serotype3 Spn, we postulated the potential use of this purified protein as atherapeutic agent for hyper virulent serotype 3 infections. Here, weinvestigated the ability of Pn3Pase to degrade the capsule on a livevirulent type 3 Spn strain and therefore render the bacteriumsusceptible to host immune clearance.

Materials and Methods Bacterial Strains and Growth Conditions

Streptococcus pneumoniae type 3 (WU2 strain) and acapsular derivative(JD908) (60, 61), generous gifts from Moon Nahm (University of Alabamaat Birmingham), were cultured aerobically without shaking at 37° C. onTryptic Soy Agar with 5% sheep blood (TSAB), or in Todd Hewitt Brothplus 0.5% yeast extract (THY) (BD Biosciences).

Mice

Eight-week-old female BALB/c mice were obtained from Taconic Biosciences(Hudson, NY) and housed in the Central Animal Facility at the Universityof Georgia. Mice were kept in microisolator cages and handled underBSL-2 hoods.

Production of Recombinant Pn3Pase

Pn3Pase was produced as described previously with minor modifications(26). Briefly, BL21(DE3) cells transformed with the pET-DEST42-“Pn3Pase”plasmid were grown in Terrific Broth supplemented with 100 μg/mlampicillin at 37° C., and cell density was monitored by absorbance at600 nm. Once the OD 600 nm reached 1.0, the cells were transferred into18° C. Protein expression was induced by the addition of Isopropylβ-D-1-thiogalactopyranoside to a final concentration of 1 mM and thecell culture was allowed to incubate for 18 h. Cells were harvested bycentrifugation, resuspended in phosphate-buffered saline (PBS, pH 7.2)and lysed by pressure lysis. The lysate was clarified by centrifugationat 17,000×g for 1 h at 4° C., and passed through a 0.45 μm syringefilter. Recombinant Pn3Pase was purified by Ni2+-NTA resin at 4° C.,eluted with 300 mM imidazole and buffer exchanged into PBS pH 7.2.Protein concentration was determined by the bicinchoninic acid assayaccording to the instructions of the manufacturer. Purity was evaluatedby coomassie staining.

Pn3Pase Treatment of Type 3 S. pneumoniae

Fresh Streptococcus pneumoniae type 3 (WU2) and acapsular derivative(JD908) colonies on TSAB plates were inoculated to 0.1 OD 600 nm in THYbroth and cultured as described above. WU2 growth in presence of 100μg/ml Pn3Pase was monitored over the course of 10 h by measuring OD 600nm. WU2 was grown in presence of 2 μg/ml, 10 μg/ml, or 10 μg/ml heatinactivated Pn3Pase for 6 hours, serially diluted, and plated todetermine colony forming units. Cultures were harvested bycentrifugation, washed in PBS, fixed in 2% paraformaldehyde for 20 minon ice, washed once more, and suspended in 1 ml PBS. For the time courseexperiment, WU2 and the acapsular strain were grown to mid-log phase (OD600 nm of 0.6), harvested by centrifugation, washed in PBS, and thensuspended in 1 ml PBS. Then, 1 μg/ml Pn3Pase was added and incubated at37° C. for the 1, 2, or 4 hours. Treated cells were serially diluted,and plated to determine colony forming units, fixed in 2%paraformaldehyde for 20 min on ice, washed once more, and suspended in 1ml PBS.

Competition ELISA

ELISA plates (96 well, Nunc) were coated with 5 μg/ml Pn3P in 0.1 Mcarbonate buffer (pH 9.0) overnight at room temperature. Plates werewashed 4-times with PBS+0.1% Tween (PBS-T) 20 using a Biotek 405/LSmicroplate washer. After 1 h blocking at room temperature with 1% BSA inPBS, microplate wells were incubated for 2 h at room temperature withfixed, treated cells that were pre-incubated for 30 min with Pn3Pspecific anti-serum in PBS-T. Plates were washed, and then incubated for2 h at room temperature with 1:2000 dilution of goat anti-mouse IgG-AP(Southern Biotech #1030-04) in PBS-T. After washing, plates wereincubated for ˜30 min at 37° C. with 2 mg/ml phosphatase substrate(Sigma S0942) in 1 M Tris 0.3 mM MgCl₂. Absorbance at 405 nm wasmeasured on a Biotek synergy H1 microplate reader. Percent inhibition ofantibody binding was calculated by((Uninhibited_(OD405)−Inhibited_(OD405))/Uninhibited_(OD405))×100.

Electron Microscopy

Electron microscopy was performed by the Georgia Electron Microscopycore facility at the University of Georgia according to a modifiedmethod by Hammerschmidt et al. (62). Treated cells were fixed in 2%glutaraldehyde, 2% paraformaldehyde, 0.075M lysine-acetate and 0.075%ruthenium red in PBS buffer for 1 h on ice and then rinsed 2× withbuffer containing 0.15% ruthenium red, 15 min per rinse. Cells were thenfixed in 1% osmium tetroxide in buffer containing 0.15% ruthenium redfor 1 h at room temperature followed by two rinses in buffer containing0.15% ruthenium red, 15 min per rinse. Pellet was dehydrated in a gradedethanol series (30%, 50%, 75%, 95%, 100% and 100%) and two changes in100% acetone, 15 min each step. Pellet was then infiltrated with 25%Spurr's resin and 75% acetone—2 h followed by sequential infiltrationwith 50% Spurr's resin and 50% acetone, 75% Spurr's resin and 25%acetone, 100% Spurr's resin and then polymerized in a 70 □C oven for 24h. Samples were sectioned at 60 nm with a Diatome diamond knife andpicked up on slot grids. Grids were post-stained on drops of uranylacetate and lead citrate, 5 min each and rinsed with H2O 30 s betweenstains. Samples were scoped using a JEOL JEM 1011 TEM (JEOL USA,Peabody, MA) operated at 80 kV.

Phagocytosis

Mid-log phase WU2 and JD908 (acapsular) cultures were washed and stainedwith 10 μM Carboxyfluorescein succinimidlyl ester (CFSE) for 30 minutesat room temperature. The WU2 strain was concurrently treated with 2μg/ml of active or heat-inactivated Pn3Pase. Bacterial cell pellets werewashed extensively, suspended in 1 ml sterile PBS. 10⁷ bacteria wereadded to a confluent monolayer of murine leukemia virus transformedmacrophage line, RAW 264.7 (American Type Culture Collection (ATCC)Manassas, VA) with active or heat-inactivated baby rabbit complement(Pel-Freez) in a 24 well plate and incubated for 1 hour at 37° C. Wellswere washed 4× with PBS to remove extracellular bacteria. Macrophageswere fixed with 2% paraformaldehyde at 4° C. for 15 minutes and removedfrom the plate. For microscopy, cells were incubated at room temperaturefor 30 minutes with a 1/500 dilution of biotinylated wheat germagglutinin (Vector labs) in 1% bovine serum albumin followed by a30-minute room temperature incubation with a 1/1000 dilution ofstreptavidin APC (Biolegend). Cells were imaged with a 40× objectivelens. Flow cytometry was performed on a Beckman Cytoflex S cytometer andanalyzed by FlowJo. Cells were gated on the macrophage population byscatter plot. A non-CFSE labeled control served to gate highlyfluorescent cell populations. The Pn3Pase dose dependent experiment wasperformed as above with addition of active complement.

Complement Deposition

A complement deposition assay was performed as described previously(63). Type 3 WU2 and acapsular (JD908) strains of bacteria wereresuspended in 3% BSA in PBS. Aliquots (in duplicate wells on a 96-wellround-bottom plate) were stained with Hoechst 33342 and treated withinactivated or functional Pn3Pase at 5 or 50 μg/ml for 1 hour at 37° C.Normal mouse serum (1:10 dilution) was added to the samples for 30minutes at 37° C. Cells were washed and stained with FITC-conjugatedgoat antibody to mouse complement (MP BioMedical, Santa Ana, CA) at 4°C. for 30 minutes. Samples were washed with 3% BSA in PBS andresuspended in 2% paraformaldehyde to fix. Samples were then analyzedwith flow cytometry. Mean fluorescent intensity (MFI) of FITC-A wascalculated from gating of Hoechst-positive cells.

Modified OPA

An opsonophagocytic killing assay was performed as described previouslywith modifications (43). Briefly, the type 3 WU2 and acapsular (JD908)strains were incubated in duplicate wells in a 96-well round-bottomplate for 1 hour or 4 hours at 37° C. with or without Pn3Pase(inactivated or functional at concentrations of 5 or 50 ug/ml) inopsonization buffer B (OBB, sterile 1×PBS with Ca++/Mg++, 0.1% gelatin,and 5% heat-inactivated FetalClone). Human promyelocytic leukemia cellline, HL-60 (ATCC Manassas, VA) were cultured in RPMI with 10%heat-inactivated FetalClone (HyClone) and 1% L-glutamine. HL-60 cellswere differentiated using 0.6% N,N-dimethylformamide (DMF, Fisher) forthree days before performing the OPA assay, harvested, and resuspendedin OBB. Active or heat-inactivated (no complement) baby rabbitcomplement (Pel-Freez) was added to HL-60 cells at 1:5 final volume. TheHL-60/complement mixture was added to the serum/bacteria at 5×10⁵cells/well (for controls, no HL-60/complement was added; equal volumesof OBB buffer was added instead). The final reactions were incubated at37° C. for 1 hour. The reactions were stopped by incubating the sampleson ice for approximately 20 minutes. Then, 10 μl of each reaction wasdiluted to a final volume of 50 μl and plated onto blood agar plates induplicate. Plates were incubated overnight at 30° C. in anaerobicconditions and counted the next day. Percent survival was calculated aseach duplicate reaction normalized to mean values obtained for controlsamples (reactions without HL-60 cells, 100% survival).

Murine Intranasal Colonization

An intranasal colonization was performed essentially as described byPuchta et al (64). Mid-log phase WU2 cultures were washed with sterilePBS and suspended at a concentration of 10⁸ CFU/ml or 10⁶ CFU/10 μl.Groups of 5 to 10 unanesthetized 8-week-old female BALB/c mice (Taconic)were intranasally inoculated with 10⁶ CFU/10 μl as previously described.Mice were either inoculated with 10 μl of PBS as vehicle on day 0, 3,and 7 or treated by administering 50 μg of Pn3Pase in 10 μl PBS on day0, 0 and 3, or 0, 3, and 7. Nasal lavage fluid was obtained on day 10 byflushing out the nasopharynx with PBS by insertion of a 25 gauge needleinto the trachea to expel 500 μl through the nares. Serial dilutions ofthe nasal lavage fluid were plated on Tryptic Soy Agar with 5% sheepblood (TSAB), to enumerate the colony forming units. A sandwich ELISA(Biolegend mouse ELISA MAX) was performed according to manufacturer'sinstructions to determine TL-6 and TNFα cytokine levels in the lavagefluid.

Murine Sepsis Challenge

Mid-log phase WU2 cultures were washed with sterile PBS and suspended ata concentration of 5×10³ CFU/100 μL. Groups of 4 unanesthetized8-week-old female BALB/c mice (Taconic) were injected intraperitoneally(I.P.) with 5×10³ CFU. Control mice were injected I.P. with 5 μg of heatinactivated Pn3Pase in 100 μL of PBS at time 0, or directly afterinfection. Treated mice were administered I.P. 0.5 μg or 5 μg of Pn3Pasein 100 μL PBS at time 0, 12, or 24 hours post infection. Animals weremonitored every 12 hours.

Results

Pn3Pase Removes Capsule from Growing Type 3 Spn

The encapsulated type 3 WU2 strain was cultured for 10 hours with therecombinant enzyme added to the growth medium and bacterial growth wasmonitored by measuring OD at 600 nm to assess effects of Pn3Pasetreatment on growing type 3 Spn. Pn3Pase treatment of cells demonstratedno adverse growth or cytotoxic effects on the bacteria (FIG. 9A). In thesame experiment, we obtained comparable CFU values for both enzymetreated and non-treated groups at two-hour intervals (data not shown).To assess direct impact of enzyme treatment on bacterial survival, alog-phase culture was isolated and suspended in nutrient-free bufferwith active, or heat inactivated Pn3Pase. Enzyme treated cells showedcomparable counts to inactivated and PBS controls over the 8-hour timecourse (FIG. 9B).

The bacterial cells grown in the presence of Pn3Pase were then examinedby competition ELISA to determine if the enzyme led to efficient capsuleremoval in the growing cultures. After treatment, fixed whole cells, attwo different concentrations, were used to compete for Pn3P specificantibodies binding to the Pn3P coated plate. The acapsular WU2 mutantstrain (JD908) showed minimal to no inhibition of antibody binding dueto its lack of capsule. Heat inactivated Pn3Pase treated cellsdemonstrated the highest percent inhibition as cell surfaces should befully decorated with CPS. With active Pn3Pase treatment, inhibition ofantibody binding begins to decrease in a Pn3Pase concentration dependentmanner (FIG. 10A), suggesting that the enzyme strips the capsule fromlive, growing type 3 Spn.

A time course experiment was performed in which the bacterial cells weregrown to mid-log phase, suspended in PBS, and treated with Pn3Pase for0, 1, 2, or 4 hours. The Pn3Pase dose was lowered to 1 μg/ml for thisassay given that the WU2 strain should not be actively growing andtherefore not producing a substantial amount of new CPS under theseconditions. At each time point, the treated cells were fixed andexamined by competition ELISA as described above. The acapsular WU2mutant strain (JD908), again, showed minimal to no inhibition due to itslack of capsule. Untreated cells exhibited the highest percentinhibition, indicative of a cell surface fully decorated with CPS. Withincreasing Pn3Pase incubation time, inhibition of antibody bindingbegins to decrease significantly, appearing essentially acapsular after2- or 4-hour treatments (FIG. 10B).

The Pn3Pase treated cells were visualized by transmission electronmicroscopy and compared to the cells treated with heat inactivatedPn3Pase. The heat inactivated enzyme treated cells displayed a thick,complete CPS coat across their surface (FIG. 10C), undoubtedly distinctfrom the acapsular mutant (FIG. 10D). The capsule layer of the enzymetreated cells exhibited little to no capsule (FIG. 10E), appearing asacapsular.

Pn3Pase Treatment of Type 3 Spn Allows Phagocytic Cell Uptake andKilling

A major virulence mechanism of the CPS is to provide the bacterium theability to resist phagocytosis by host phagocytic cells (1, 6). Toinvestigate the effect of Pn3Pase treatment on uptake by macrophages invitro, we stained mid-log phase bacterial cultures withcarboxyfluorescein succinimidlyl ester (CFSE) and then treated withPn3Pase followed by incubation with RAW 264.7 macrophages. Macrophageswere then washed extensively and imaged by fluorescent microscopy (FIG.11A). The extent of bacterial uptake by macrophages was quantified byflow cytometry. Bacteria treated with the enzyme were taken up bymacrophages significantly more than the encapsulated strain. We thendetermined the percentage of fluorescent macrophages, representing thephagocytosis of fluorescent bacteria (FIGS. 11C-D). The encapsulatedtype 3 strain incubated with heat inactivated Pn3Pase had minimalfluorescently labeled phagocytes whereas Pn3Pase treated bacteria wereas efficiently taken up by the macrophages as the acapsular mutantstrain. Uptake was partially dependent on complement as evidenced byhigher bacterial uptake in the presence of complement (FIG. 11C).Pn3Pase treatment rendered the bacteria more susceptible to phagocyticengulfment by RAW macrophages in a dose dependent manner, while evenhigh doses of the inactivated enzyme had no significant effect onbacterial uptake (FIG. 11D).

Complement deposition on the pneumococcal surface is an importantmechanism aiding in efficient phagocytosis and clearance (10, 11). SinceCPS provides complement evasive properties to the bacterium (10, 11), weinvestigated the effect of capsule removal by Pn3Pase treatment on C3bdeposition on the bacterial surface. The encapsulated type 3 WU2 strainwas treated with active or inactive Pn3Pase. The untreated type 3 strainand the acapsular mutant were included as additional controls. Bacteriawere then incubated with normal mouse serum, washed, and stained withFITC-conjugated antibody to mouse complement. Fixed samples were thenanalyzed with flow cytometry. In a dose dependent manner, Pn3Pasetreatment increased the deposition of complement on the bacterialsurface, reaching the levels of deposition on the acapsular strain.Complement had minimal binding to the bacteria either untreated ortreated with inactivated-Pn3Pase (FIG. 12 ).

The standard in vitro assay to assess phagocytic killing of Spn is anopsonophagocytosis assay (OPA) in which HL-60 cells are differentiatedinto neutrophils to engulf and clear antibody opsonized bacteria. Thismethod has been widely used to measure the quality of antibody responsesin numerous vaccine studies (43-48). Neutrophils are one of the mostimportant components of innate immunity against pathogenic bacteria inthe lungs (49-51). While the focus of this study is not on humoralimmune responses to Spn, we have used a modified OPA to evaluatecomplement-mediated neutrophil killing of type 3 Spn treated withPn3Pase in vitro. Encapsulated type 3 Spn was incubated with active orheat inactivated Pn3Pase. Differentiated HL-60 cells were pre-incubatedwith active or heat-inactivated complement. The mixture of HL-60 cellsand complement was added to the bacteria and incubated at 37° C. for 1hour. To quantify the surviving bacteria in the experimental groups, thereaction mixtures were plated and CFUs were counted the next day.Percent survival was calculated as each duplicate reaction normalized tomean values obtained from reactions without neutrophils, which served ascontrol samples (100% survival). The enzyme-untreated, encapsulated type3 strain was able to escape neutrophil killing to show maximum survivalwhile the acapsular mutant strain was reduced to nearly 40% viabilityupon incubation with active complement and neutrophils (FIG. 13 ).Pn3Pase treatment to remove the capsule rendered the type 3 strainsusceptible to complement dependent neutrophil killing similar to theacapsular strain (FIG. 13 ).

Pn3Pase Limits Nasopharyngeal Colonization

To investigate the enzyme's protective abilities in vivo we firstperformed an intranasal colonization experiment with BALB/c mice. Nasalcolonization by Spn is essential for transition to invasive pneumococcaldisease (4, 5). It is established that the capsule of this strain isrequired for intranasal colonization (6). Therefore, we used the nasalcolonization model to assess the ability of Pn3Pase to reduce bacterialcolonization in the nasopharynx through removal of the capsule of thecolonizing type 3 strain. We first confirmed that the acapsular mutant,JD908, failed to colonize the nasopharynx (data not shown). Groups ofmice were then intranasally inoculated with 10⁶ log-phase wildtype (wt)encapsulated bacteria in 101 PBS. All inocula were chased with eitherPn3Pase or buffer control. Groups were dosed with the enzyme at eitherday 0, day 0 and 3, or day 0, 3, and 7 to assess the effects of multipleadministrations. Mice were euthanized, and bacterial load was quantifiedon day 10. Nasal lavage fluid was obtained, serially diluted, and platedto enumerate the bacterial load. Vehicle control treated mice werecolonized with significantly higher bacterial loads than mice treatedwith only a single dose of Pn3Pase. Administration of two or three dosesof Pn3Pase made the majority of the animal lavage fluid void of anyviable bacterial colonies (FIG. 14A). Lung homogenates and serum samplesshowed no evidence of a bacterial burden (data not shown). Signaturepro-inflammatory cytokine levels were measured in the nasal lavage fluidby ELISA (52). Vehicle treated mice had significantly increased levelsof the cytokines IL-6 and TNFα compared to Pn3Pase treated animals, areflection of a continued host inflammatory response to the bacterialburden in this group (FIGS. 14B-C) (52). A significant reduction in IL-6and TNFα was observed in most animals even after a single dose ofPn3Pase on day 0. The mouse with higher bacterial load in the singledose group contributed to the increased cytokine levels in this group.

Pn3Pase Protects Mice from Lethal Challenge

To further assess Pn3Pase for its protective abilities and evaluate theutility of Pn3Pase as a therapeutic agent, we employed anintraperitoneal (I.P.) sepsis model (53, 54). Groups of mice wereinfected with 5×10³ CFU of log-phase WU2 type 3 strain of Spn. Weassessed the effect of a single dose of 5 g or 0.5 g, administered attime 0, 12, or 24 hours post infection. Control groups treated with heatinactivated enzyme died within 48 hours of infection. Regardless of theenzyme dose or the timing of the administration, all treated groupsdisplayed no signs of illness and experienced full protection from theI.P. challenge (FIG. 15 ).

Discussion

This study aimed to evaluate the protective role of acarbohydrate-degrading enzyme (glycoside hydrolase), Pn3Pase, targetingthe CPS of the pathogenic bacterium, serotype 3 Spn. Invasivepneumococcal diseases (IPD) caused by Spn have been a major threat tohuman health with alarming mortality rates. Despite a global vaccinationprogram and the use of antibiotics, Spn remains among the deadliestinfectious agents worldwide. Pneumococcal vaccines are made empiricallyand are variably/poorly immunogenic, especially among elderly andimmunocompromised individuals. Widespread use of antibiotics against IPDhas led to spread of drug resistant pneumococcal strains (34, 35). Thisstudy offers an alternative targeted therapeutic approach to theshortcomings of the incumbent vaccine and antibiotic solutions to IPD.

First we demonstrated that Pn3Pase could efficiently remove the capsulefrom live pneumococci without having bactericidal effects on the cells.Through in vitro assays we observed that Pn3Pase treatment increases thebacterium's susceptibility to phagocytosis by a macrophage cell line.These results were promising since the capsule is a major host immuneevasion component that allows Spn to resist engulfment by hostphagocytes (9). We then concluded that enzyme treatment significantlyincreased complement-mediated killing by the neutrophils. A single doseof Pn3Pase reduced murine nasopharyngeal colonization by type 3 Spnsignificantly, indicating that the enzyme may function as a prophylacticmeasure to control colonization by this serotype in at-risk populations.Finally, an intraperitoneal challenge was performed to assess theprotective capacity of Pn3Pase in a sepsis model. Notably, a single lowdose of 0.5 μg administered 24 hours after infection was able to protect100% of the challenged mice from the bacterial challenge while controltreated animals did not survive longer than 48 hours. The robustprotective capacity of Pn3Pase in this model demonstrates the enzymaticactivity is sufficient within the host to effectively degrade thecapsule even at low doses.

Given that Pn3Pase has therapeutic potential for pneumococcalinfections, practical issues pertaining to the application of the enzymesuch as immunogenicity, administration routes, and substrate specificitywill need to be addressed (55). Our preliminary assessment of antibodytiters generated against Pn3Pase in the challenge experiments observedno IgM or IgG response generated against the effective dose of theenzyme. Future studies will evaluate humoral and cellular immuneresponses to Pn3Pase and investigate alternative routes ofadministration such as intravenous for a bacteremia model, oraerosolized spray for the pneumonia model. A useful example of enzymedelivery to the respiratory tract by aerosol spray is the recombinanthuman deoxyribonuclease known as Pulmozyme, used to relieve airwayobstruction by secreted DNA in cystic fibrosis patients (56). Anotherpotential problem with the use of Pn3Pase as a therapeutic agent is itsactivity on host glycans. Relaxed substrate specificity of Pn3Pase onmammalian glycans structurally similar to Pn3P will need to be assessed.

In addition to the high potential of Pn3Pase as a therapeutic enzyme,this glycosyl hydrolase has unique properties from a structure/functionpoint of view in that it does not fall into a currently establishedglycosyl hydrolase Carbohydrate Active enZYme (CAZY) family (57).Further examination of structural properties of this protein may lead tothe discovery of structurally similar enzymes with activities towardother unique bacterial CPSs. Future investigations will explore theexistence of enzymes for use against other prevalent pneumococcalserotypes and other encapsulated pathogenic bacteria. Based on earlierstudies, the native species expressing Pn3Pase has the capacity todegrade two additional pneumococcal CPSs (58, 59). While this Pn3Paseexpressing Paenibacillus species was isolated from the soil (26, 42,59), it is befitting to question why this species would evolve topossess such enzymes that are capable of degrading capsules of a humanpathogen. Whether these are the natural substrates for the enzymes,indicating co-evolutionary relationship, or whether other soil-dwellingmicrobes or plants express similar glycan residues and linkages remainsto be explored.

The results presented here indicate that enzymatic hydrolysis of the CPSmay be a valid alternative or complementary therapeutic approach fordiseases caused by Spn and potentially other important encapsulatedpathogens such as Neisseria meningitidis and Methicillin-ResistantStaphylococcus aureus (MRSA). In summary, this study serves as the firstcomprehensive evaluation of the protective role of a glycosidehydrolase, Pn3Pase, debilitating an otherwise lethal bacterial pathogenthrough targeting its capsular polysaccharide.

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The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference in their entirety.Supplementary materials referenced in publications (such assupplementary tables, supplementary figures, supplementary materials andmethods, and/or supplementary experimental data) are likewiseincorporated by reference in their entirety. In the event that anyinconsistency exists between the disclosure of the present applicationand the disclosure(s) of any document incorporated herein by reference,the disclosure of the present application shall govern. The foregoingdetailed description and examples have been given for clarity ofunderstanding only. No unnecessary limitations are to be understoodtherefrom. The disclosure is not limited to the exact details shown anddescribed, for variations obvious to one skilled in the art will beincluded within the disclosure defined by the claims.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless otherwise indicated to thecontrary, the numerical parameters set forth in the specification andclaims are approximations that may vary depending upon the desiredproperties sought to be obtained by the present disclosure. At the veryleast, and not as an attempt to limit the doctrine of equivalents to thescope of the claims, each numerical parameter should at least beconstrued in light of the number of reported significant digits and byapplying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the disclosure are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. All numerical values, however, inherently contain a rangenecessarily resulting from the standard deviation found in theirrespective testing measurements.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

1.-50. (canceled)
 51. A genetically modified cell comprising a maturePn3Pase protein, wherein the protein has Pn3Pase activity.
 52. Agenetically modified cell comprising a polynucleotide comprising acoding region, wherein the coding region comprises a nucleotide sequenceencoding a mature Pn3Pase protein, and wherein the protein has Pn3Paseactivity.
 53. The genetically modified cell of claim 51 wherein theprotein has at least 80% identity with the amino acid sequence set forthin SEQ ID NO:2, wherein the amino terminal amino acid is selected fromany one of residues 2 to 64 of SEQ ID NO:2 and the carboxy terminalamino acid is residue 1545 of SEQ ID NO:2.
 54. The genetically modifiedcell of claim 53 wherein the protein has at least 80% identity withamino acids 41-1545 of SEQ ID NO:2.
 55. The genetically modified cell ofclaim 51 wherein the cell is a eukaryotic cell.
 56. The geneticallymodified cell of claim 55 wherein the cell is a mammalian cell, a yeastcell, or an insect cell.
 57. The genetically modified cell of claim 51wherein the cell is a prokaryotic cell.
 58. The genetically modifiedcell of claim 57 wherein the cell is E. coli.
 59. The geneticallymodified cell of claim 51 wherein the protein comprises a heterologousamino acid sequence.
 60. The genetically modified cell of claim 59wherein the heterologous amino acid sequence comprises a tag.
 61. Acomposition comprising the genetically modified cell of claim
 51. 62.The genetically modified cell of claim 51, wherein the cell comprises anexogenous polynucleotide comprising a coding region encoding a Pn3Paseprotein.
 63. The genetically modified cell of claim 62, wherein theexogenous polynucleotide comprises an expression vector.
 64. Thegenetically modified cell of claim 62, wherein the exogenouspolynucleotide comprises a marker sequence.
 65. The genetically modifiedcell of claim 64, wherein the marker sequence renders the geneticallymodified cell resistant to an antibiotic.
 66. A method comprisingincubating the genetically modified cell of claim 54 under suitableconditions for expression of a Pn3Pase protein.
 67. The method of claim66, wherein suitable conditions comprise Pn3P.
 68. The method of claim66, further comprising isolating the Pn3Pase protein from the cell ormedium.
 69. The method of claim 68, wherein isolating the Pn3Paseprotein from the cell comprises lysing the cell.
 70. The method of claim68, wherein isolating the Pn3Pase protein comprises purifying thePn3Pase protein.